SOURCES AND EFFECTS
OF IONIZING RADIATION
United Nations Scientific Committee on the Effectsof Atomic Radiation
UNSCEAR 2000 Report to the General Assembly,
with Scientific Annexes
VOLUME II: EFFECTS

UNITED NATIONSNew York, 2000

NOTE
The report of the Committee without its annexes appears as Official Records of theGeneral Assembly, Fifty-fifth Session, Supplement No. 46 (A/55/46).
The designation employed and the presentation of material in this publication do not
imply the expression of any opinion whatsoever on the part of the Secretariat of the United
Nations concerning the legal status of any country, territory, city or area, or of its authorities,
or concerning the delimitation of its frontiers or boundaries.
The country names used in this document are, in most cases, those that were in use
at the time the data were collected or the text prepared. In other cases, however, the names
have been updated, where this was possible and appropriate, to reflect political changes.

UNITED NATIONS PUBLICATION
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ANNEX F
DNA repair and mutagenesis

CONTENTSPage

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
I.

DNA DAMAGE AND REPAIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. THE ROLE OF DNA REPAIR GENES IN CELL FUNCTION . . . . . . . . . .
B. TYPES OF DAMAGE AND PATHWAYS OF REPAIR . . . . . . . . . . . . . . .
C. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2
2
3
8

II.

REPAIR PROCESSES AND RADIOSENSITIVITY . . . . . . . . . . . . . . . . . . . . . . 9
A. RADIOSENSITIVITY IN MAMMALIAN CELLS AND HUMANS . . . . . . 9
1. The identification of radiosensitive cell lines and disorders . . . . . . . . . 9
2. Mechanisms of enhanced sensitivity in human disorders . . . . . . . . . . 11
3. Analysis of genes determining radiosensitivity . . . . . . . . . . . . . . . . . . 12
4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
B. RELATIONSHIP BETWEEN REPAIR AND
OTHER CELL REGULATORY PROCESSES . . . . . . . . . . . . . . . . . . . . . . 18
1. Radiosensitivity and defective recombination in the immune system:
non-homologous end joining of DNA double-strand breaks . . . . . . . . 18
2. Radiosensitivity and the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3. Apoptosis: an alternative to repair? . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

III. HUMAN RADIATION RESPONSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. CONTRIBUTION OF MUTANT GENESTO HUMAN RADIOSENSITIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. INFLUENCE OF REPAIR ON RADIATION RESPONSES . . . . . . . . . . . .
C. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26
26
29
32

IV. MECHANISMS OF RADIATION MUTAGENESIS . . . . . . . . . . . . . . . . . . . . .
A. MUTATION AS A REPAIR-RELATED RESPONSE . . . . . . . . . . . . . . . .
B. THE SPECTRUM OF RADIATION-INDUCED MUTATIONS . . . . . . . . .
C. MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATIONS .
D. EFFECT OF RADIATION QUALITY . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. NOVEL MECHANISMS OF GENETIC CHANGE . . . . . . . . . . . . . . . . . .
F. MUTATION FREQUENCIES AND CONSEQUENCES . . . . . . . . . . . . . .
G. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33
33
35
36
39
41
44
47

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

2

ANNEX F: DNA REPAIR AND MUTAGENESISINTRODUCTION
1. Risk estimates for the induction of human disease are
obtained primarilyfrom epidemiological studies. Thesestudies
can clearly distinguish radiation effects only at relatively highdoses and dose rates. Togain information at low doses and low
dose rates, which are more relevant to typical human radiation
exposures, it is necessary to extrapolate the results of thesestudies. To be valid, this extrapolation requires a detailed
understanding of the mechanisms by which radiation induces
cancer and genetic disorders.
2. Several lines of evidence show that sites of radiationinduced cell lethality, mutation, and malignant change aresituated within the nucleus and that DNA is the primarytarget. When DNA is damaged by radiation, enzymes within
the cell nucleus attempt to repair that damage. The efficiencyof the enzymatic repair processes determines the outcome:most commonly, the structure of DNA is repaired correctly
and cellular functions return to normal. If the repair is
unsuccessful, incomplete, or imprecise, the cell may die or
may suffer alteration and loss of genetic information (seen as
mutation and chromosomal aberration). These information
changes determine heritable genetic defects and are thought
to be important in the development of radiation-induced
cancer. The more complete the knowledge of the ways inwhich human cells respond to damage and of the mechanisms
underlying the formation of mutations and chromosomal
aberrations, the more accurate will be the predictions of theoncogenic and hereditary effects of ionizing radiation.

3.
DNA repair is itself controlled by a specific set ofgenes encoding the enzymes that catalyse cellular response
to DNA damage. Loss of repair function, or alteration of
the control of repair processes, can have very serious
consequences for cells and individuals. It is anticipated that
DNA repair plays a critical role in protecting normal
individuals from radiation effects, including cancer.
Clinical experience has revealed individuals who are both
hypersensitive to radiation and cancer-prone; some of these
individuals have recently been shown to have defects ingenes involved in the response to DNA damage.
4.
In recent years there have been significant advancesin the molecular analysis of repair processes and theunderstanding of the mechanisms that induce genetic
changes. Additionally, new methods have been developed
to simplify the identification of the genes involved. As thedetails of damage-repair processes become clearer, it isseen that these processes have considerable overlap withother cellular control functions, such as those regulating
the cell cycle and immune defences. In this Annex theCommittee continues to review such developments inmolecular radiobiology, as it began to do in Annex E,“Mechanisms of radiation carcinogenesis” of the
UNSCEAR 1993 Report [U3], in order to improve theunderstanding of how radiation effects are manifested incells and organisms.

I. DNA DAMAGE AND REPAIR
A. THE ROLE OF DNA REPAIR GENESIN CELL FUNCTION
5. The information needed to control cellular functions
such as growth, division, and differentiation is carried by
the genes. Genes, which are specific sequences of DNA, act
mainlythrough the production of complementarymessages(mRNA) that are translated into proteins. Proteins canhave a structural role but commonly work as enzymes, eachof which catalyses a particular metabolic reaction. Thus
specific genes contain the code for (encode) specificcellular functions. The production of proteins can be timed
so that they work at specific points in the development ofa cell or organism, but protein function can also becontrolled by post-translational modifications. These
modifications are carried out by other proteins, so that acomplex set of interactions is necessary to fine-tune
cellular functions. Proteins involved in important aspectsof cell metabolism (e.g. DNA replication) may also workin multi-protein complexes [A1]. There is some evidence
also that some of the complexes are assembled into larger
structures situated in defined regions of the nucleus (e.g.
the nuclear matrix) [H2].

6.
Loss or alteration of information in a specific gene may
mean that none of that gene product (protein) is formed, orthat the protein is less active, or that it is formed in an
uncontrolled fashion (e.g. at the wrong time or in the wrong
amount). While some minor genetic alterations may not affectprotein activity or interactions, others may significantly
disrupt cellular function. Since certain proteins work in a
number of different processes or complexes, the loss or
impairment of one type of protein can affect several different
functions of the cell and organism (pleiotropic effect).
7.
A verylarge number of genes, 60,000-70,000 [F14], arerequired to control the normal functions of mammalian cells
and organisms. However, the genes form only a small part of
the genome (the complete DNA sequence of an organism), theremainder of which largely consists of many copies ofrepetetive DNA sequence. The genes are linked in linear
arrays interspersed by non-coding sequences, to form
chromosomes located in the cell nucleus. Most genes arepresent in only two copies, each on a separate homologous
chromosome, one inherited from the mother and one from thefather. To monitor damage and to maintain the genes without
significant alteration is a major concern for the cell. Repair

ANNEX F: DNA REPAIR AND MUTAGENESIS

3

processes are common to all organisms from bacteria tohumans and have evolved to correct errors made in replicating
the genes and to restore damaged DNA. This fact has inrecent years provided a useful tool for molecular geneticists inthe analysis of repair processes; well characterized microorganisms can serve as model systems to understand thestructure and function of repair genes. The information gainedin this way can sometimes also be used directly to isolate
human genes of related function [L1]. While the structure andfunction of repair genes appears to be highly conserved fromlower to higher organisms, the regulation of their activities
may differ in different organisms.

are arranged in two linear arrays (or strands) held together byhydrogen bonds centrally and linked externally by covalentbonds to sugar-phosphate residues (the DNA "backbone").
The adenine base pairs naturally with thymine (A:T basepair), while guanine pairs with cytosine (G:C base pair), sothat one DNA strand has the complementary sequence of theother. The sequence of the bases defines the genetic code; eachgene has a unique sequence, although certain common
sequences exist in control and structural DNA elements.
Damage to DNA may affect any one of its components, but itis the loss or alteration of base sequence that has genetic
consequences.

8. The consequences of loss of repair capacity are seen ina number of human syndromes and mutant cell lines. These
showhypersensitivitytoenvironmental agents, and the humansyndromes often have multiple symptoms, including cancerproneness, neurological disorder, and immune dysfunction.
Good progress has been made over the last few years inmapping and cloning the genes involved.

12. Ionizing radiation deposits energy in tracks ofionizations from moving charged particles within cells, andradiations of different quality may be arbitrarily divided into
sparsely ionizing, or low linear energy transfer (low-LET),
and densely ionizing (high-LET). Each track of low-LET
radiations, such as x rays or gamma rays, consists of only arelatively small number of ionizations across an average-sizedcell nucleus (e.g. a gamma-ray electron track crossing an
8 µm diameter nucleus gives an average of about 70
ionizations, equivalent to about 1 mGy absorbed dose,
although individual tracks vary widely about this value
because of track stochastics and varying path lengths throughthe nucleus). Each track of a high-LET radiation may consistof many thousands of ionizations and give a relatively highdose to the cell; for example, a 4 MeV alpha-particle track
has, on average, about 23,000 ionizations (370 mGy) in an
average-sized cell nucleus [G27, U3]. However, within the
nucleus even low-LET radiations will give some small regionsof relatively dense ionization over the dimensions of DNA
structures, for example, where a low-energy secondary
electron comes to rest within a cell.

9. Ionizing radiation damages DNA and causes mutationand chromosomal changes in cells and organisms. Damage byradiation or radiomimetic agents also leads to cell transformation (a stage in cancer development) and cell death. In thelight of current research, it is seen that the final response toradiation damage is determined not only by cellular repair
processes but also by related cellular functions that optimize
the opportunity for recovery from damage. For example,
radiation damage may cause an arrest in the cell cycle; this isthought to be a damage-limitation step, allowing time forrepair and reducing the consequences of a given dose [L22].
There is now some understanding of the way in whichradiation alters cell cycle timing (Section II.B.2), although theroles of a number of enzymatic activities that are induced or
repressed shortly after irradiation remain to be clarified(Section III.B).
10. The severity of DNA damage, or the context in which
damage occurs (e.g. during DNA replication), will often
dictate a repair strategy that places survival first and incurs
genetic change. DNA replication may bypass sites of singlestranded DNA damage, inserting an incorrect base opposite
the altered or lost base. Additionally, in attempting to repair
damage to DNA, enzymes may not be able to restore thestructure with fidelity. Thus, mutation and chromosomal
rearrangement are not passive responses to damage; rather,
they are a consequence of the interaction of cellular processes
with damage. The types of genetic change that occur will
depend on the types of initial DNA damage, from theirpotential to miscode at replication and from the probability
that specific repair enzymes will act on given types of damage.

B. TYPES OF DAMAGE AND PATHWAYS
OF REPAIR
11. DNA is a double-helical macromolecule consisting offour units: the purine bases adenine (A) and guanine (G), andthe pyrimidine bases thymine (T) and cytosine (C). The bases

13. Radiation tracks may deposit energy directly in DNA(direct effect) or may ionize other molecules closely
associated with DNA, especially water, to form free radicals
that can damage DNA (indirect effect). Within a cell theindirect effect occurs over very short distances, of the order ofa few nanometres, because the diffusion distance of radicals is
limited by their reactivity. Although it is difficult to measureaccurately the different contributions made by the direct andindirect effects to DNA damage caused by low-LET radiation,evidence from radical scavengers introduced into cells
suggests that about 35% is exclusively direct and 65% has anindirect (scavengeable) component [R21]. It has been argued
that both direct and indirect effects cause similar earlydamage
to DNA; this is because the ion radicals produced by direct
ionization of DNA may react further to produce DNA
radicals similar to those produced by water-radical attack on
DNA [W43].
14. Ionization will frequently disrupt chemical bonding incellular molecules such as DNA, but where the majority ofionizations occur as single isolated events (low-LET
radiations), these disruptions will be readily repaired bycellular enzymes. However, the average density of ionization
by high-LET radiations is such that several ionizations arelikely to occur as the particle traverses a DNA double helix.

4

ANNEX F: DNA REPAIR AND MUTAGENESIS

Therefore, much of the damage from high-LET radiations, aswell as a minority of the DNA damage from low-LET
radiations, will derive from localized clusters of ionizations
that can severely disrupt the DNA structure [G27, W44].
While the extent of local clustering of ionizations in DNA
from single tracks of low- and high-LET radiations will
overlap, high-LET radiation tracks are more efficient at
inducing larger clusters, and hence more complex damage.
Also, high-LET radiations will induce some very large
clusters of ionizations that do not occur with low-LET
radiations; the resulting damage may be irrepairable but may
also have unique cellular consequences (see paras. 192, 199,
and 201) [G28]. Additionally, when a cell is damaged byhigh-LET radiation, each track will give large numbers ofionizations, so that the cell will receive a relatively high dose
and there will be a greater probability of correlated damage
within a single DNA molecule (or chromosome) or in separate
chromosomes. As a consequence, the irradiation of a
population of cells or a tissue with a “low dose” of high-LET
radiation results in a few cells being hit with a relatively highdose (one track) rather than in each cell receiving a small
dose. In contrast, low-LET radiation is more uniformly
distributed over the cell population; at doses of low-LET
radiation in excess of about 1 mGy (for an average-size cell
nucleus of 8 µm diameter), each cell nucleus is likely to be
traversed by more than one sparsely-ionizing track.
15. The interaction of ionizing radiation with DNA
produces numerous types of damage; the chemical products ofmany of these have been identified and classified according totheir structure [H4, S3]. These products differ according towhich chemical bond is attacked, which base is modified, andthe extent of the damage within a given segment of DNA.
Table 1 lists some of the main damage products that can bemeasured following low-LET irradiation of DNA, with arough estimate of their abundance. Attempts have also been
made to predict the frequencies ofdifferent damage types froma knowledge of radiation track structure, with certain
assumptions about the minimum energy deposition (numberof ionizations) required. Interactions can be classified

according to the probability they will cause a single-strand
DNA alteration (e.g. a break in the backbone or basealteration) or alterations in both strands in close proximity in
one DNA molecule (e.g. a double-strand break), or a more
complex type of DNA damage (e.g. a double-strand breakwith adjacent damage). Good agreement has been obtained
between these predictions and direct measurements of singlestrand breaks, but there is less good agreement for othercategories of damage [C47]. While complex forms of damage
are difficult to quantify with current experimental techniques,
the use of enzymes that cut DNA at sites of base damage
suggests that irradiation of DNA in solution gives complex
damage sites consisting mainlyof closely-spaced base damage(measured as oxidised bases or abasic sites); double-strand
breaks were associated with only 20% of the complex damage
sites [S87]. It is expected that the occurrence of more complex
types of damage will increase with increasing LET, and that
this category of damage will be less repairable than thesimpler forms of damage. Theoretical simulations havepredicted that about 30% of DNA double-strand breaks fromlow-LET radiation are complex by virtue of additional breaks[N19] and that this proportion rises to more than 70%, andthe degree of complexity increases, for high-LET particles[G29].
16. Some of the DNA damage caused by ionizing
radiation is chemically similar to damage that occurs
naturally in the cell: this "spontaneous" damage arises
from the thermal instability of DNA as well as endogenous
oxidative and enzymatic processes [L2, M40]. Several
metabolic pathways generate oxidative radicals within thecell, and these radicals can attack DNA to give both DNA
base damage and breakage, mostly as isolated events[B46]. The more complex types of damage caused byionizing radiation may not occur spontaneously, sincelocalized concentrations of endogenous radicals are lesslikely to be generated in the immediate vicinity of DNA.
This theme is taken up in Annex G, “Biological effects at
low radiation doses”, which considers the cellularresponses to low doses of radiation.

Table 1
Estimated yields of DNA damage in mammalian cells caused by low-LET radiation exposure[L60, P31, W39]aType of damage

Yield (number of defects per cell Gy-1)

Single-strand breaks
Base damage a
Double-strand breaks
DNA-protein cross-links

1 000
500
40
150

Base excision enzyme-sensitive sites [P31] or antibody detection of thymine glycol [L60].

17. Measurement of the endogenous levels of DNA basedamage has been difficult because of the artefactual production of damage during the preparation of the DNA for analysis(e.g. by gas chromatography/mass spectrometry) [C55]. Thisdifficulty explains the presence in the literature of considerably inflated (by factors of at least 100) values for backgroundlevels of base damage. Interestingly, the recognition ofdamage by base excision repair enzymes (paragraph 22) has
provided a less discordant method of measurement, although
the specificity of the enzymes for different types of basedamage is not precisely known. These enzymes cut the DNA
at the site of base damage, to give a single-strand break that

ANNEX F: DNA REPAIR AND MUTAGENESIS

can be measured accurately by a number of techniques. Using
this method, measurement of an important form of oxidative
damage, 7,8-dihydro-8-oxoguanine (generally known as 8oxoguanine)), has given steady-state levels of 500-2000 per
cell, depending on cell type [P30]. Using similar measurement
methods, the level of 8-oxoguanine induced in cellular DNA
by gamma rays is about 250 per cell per Gy [P31]. A newly
developed ultrasensitive assayfor another type of base damage
in human cellular DNA, thymine glycol, couples antibody
detection with capillary electrophoresis. This method showeda linear response for yield of thymine glycol with gamma-ray
dose down to 0.05 Gy, giving a level of about 500 thymine
glycols per cell per Gy against a background of 6 thymine
glycols per cell [L60]. The difficulties experienced inmeasuring base damage accurately in cellular DNA and therelatively low levels now found for the commoner types ofdamage have also called into question the extent to whichsome previouslyidentified forms of base damage occur in cells
following irradiation.
18. The measurement of endogenous levels of other types of
DNA damage, such as double-strand breaks, has involved
similar technical difficulties. Many of the methods used tomeasure double-strand breaks in mammalian cells introduce
this form of damage either inadvertentlyor deliberately as partof the methodology. This is because the mammalian genome
is so large that it had to be reduced in size by random
breakage first before useful measurements could be made.
This problem has been overcome in part by the introductionof methods based on the gentle release of DNA from cells by
their lysis in a gel matrix [C64, O3], but there is commonly
still a background level of DNA breakage amounting to a few
per cent of the total DNA. However, as documented in Section
II.B, it is unlikely that mammalian cells have a high steadystate level of DNA double-strand breakage, since these breaks
act as a signal for damage-recognition processes that can
block the cell cycle or induce programmed cell death. It is
possible that even one unrepaired double-strand break can
trigger this cellular response (paragraph 101). It has also beenfound that one unrepaired double-strand break can cause
lethality in irradiated yeast cells (paragraph 108). Thus,
tolerance of this form of damage in cells is likely to be verylow.
19. While the precise nature of the damage will influence
repairability, it is possible to consider a few general categoriesof damage in order to describe their consequences. A
simplified classification can be based on the abilityof enzymes
to use the complementary base structure of DNA to facilitate
repair of the damage site. Thus, damage to single strands(base modifications, single-strand breaks) can be removed or
modified, followed byresynthesis using the undamaged strand
as a template. Where the damage affects both strands of a
DNA molecule in close proximity (double-strand breaks,
cross-links), it is more difficult to repair and requires different
enzymatic pathways for its resolution. To resolve successfully
more complex types of damage may require enzymes from
more than one repair pathway. To illustrate the knowledge ofthe different repair pathways available to the cell, thefollowing account (to paragraph 34) includes a discussion of5

the repair of damage caused by various DNA-damaging
agents as well as ionizing radiation.
20. DNA repair enzymes can be characterized as cellular
proteins acting directly on damaged DNA in an attempt torestore the correct DNA sequence and structure. These
relatively specialized enzymes appear to undertake theinitial stages of recognition and repair of specific forms of
DNA damage. For example, DNA glycosylases catalyze thecleavage of base-sugar bonds in DNA, acting only on
altered or damaged bases [W1]. Further, there are several
different types of glycosylase that recognize chemicallydifferent forms of base damage. However, enzymes thatcarry out normal DNA metabolism are also part of therepair process for many different forms of damage. In thelatter category there are, for example, enzymes involved in
the synthesis of DNA strands (DNA polymerases) andenzymes involved in the joining of the DNA backbone(DNA ligases). Several different types of DNA polymerases
and ligases have been identified; it is thought that they
have different roles in normal DNA metabolism and that
only some are active in DNA repair [L3, P1].
21. The simplest repair processes directly reverse thedamage; for example, many organisms, but not mammals,possess an enzyme that directly photoreactivates the UVinduced dimerization of pyrimidine bases [S1]. Similarly,
the enzyme O6methylguanine-methyltransferase directly
removes methyl groups induced in DNA by alkylating
carcinogens [P13]. However, most damage types require
the concerted action of a number of enzymes, forming a
repair pathway. Several apparently discrete repair
pathways have been identified, as described below andillustrated in Figure I.
22. Damage to individual bases in DNA may be corrected
simply by removing the base, cleaning up the site, andresynthesis. In this process, termed the base-excision repair
pathway, a DNA glycosylase removes the damaged base, a
DNA endonuclease cuts the DNA backbone, the sugarphosphate remnants are removed by a phosphodiesterase,
and a polymerase fills in the gap using the opposite base asa template (Figure Ia) [L2, S49]. Even where a single baseis damaged, therefore, several different enzymes are
required to give correct repair. The latter part of thisprocess may also be used to repair single-strand breaks in
DNA. Radiation-induced DNA breaks are generally notrejoined by a simple ligation step, because sugar damage
and, often, base loss occur at the site of a break. Baseexcision repair is generally localized to the single DNAbase and is very rapid [D16, S58]; however, in mammaliancells a minority of repair patches of up to 6 bases have beenfound, indicating a second “long-patch” pathway (seeparagraph 75).
23. Many DNA glycosylases are specific for the removalof one type of altered base from DNA; for example, uracilDNA glycosylase removes only uracil and some oxidation
products of uracil [F15]. However, there is some overlap inthe specificity of some base-excision repair enzymes. An

6

ANNEX F: DNA REPAIR AND MUTAGENESISBASE DAMAGE

Glycosylase
removal

DOUBLE-STRAND BREAK

Helicase opening

Endonuclease
incision

Endonuclease
incision

Polymerase
filling

Polymerase
filling

Ligasejoining

BASE EXCISIONREPAIR
Figure Ia.

End processing

Ligasejoining

End processing

Strand invasionof an unbroken
homologue

Polymerase
filling

Ligasejoining

Ligase joining(recombination)

NUCLEOTIDENON-HOMOLOGOUS HOMOLOGOUS
RECOMBINATIONEXCISION REPAIRENDREPAIRJOINING
Figure Ib.

Figure Ic.

Figure Id.

Figure I.
Mechanisms of DNA repair (simplified).
Figure Ia: Base damage is excised by a specific glycosylase: the DNA backbone is cut and the gap filled by apolymerase. The resulting gap is refilled.
Figure Ib: Bulky base damage is removed along with an oligonucleotide (of about 30 bases in human cells). Resynthesis
takes place using the opposite strand as a template.
Figure Ic: A double-strand break is rejoined end-to-end.
Figure Id: A double-strand break is repaired with the help of a homologous undamaged molecule (shown in red). Strand
invasion allows resynthesis on complementary sequence, followed by a resolution of the strands and rejoining.

example of this is seen in the response to ionizing radiation ofbacterial cells defective for one or more nucleases involved inbase-excision repair: while cells defective for either nuclease
alone were not radiation sensitive, the loss of both nucleases
made the cells extremelyradiation sensitive [C60, Z10]. Someglycosylases have a very broad specificity for different types ofbase structure, such as 3-methyladenine-DNA glycosylase II,
which acts on a variety of chemically-modified purines andpyrimidines, 5-methyloxidized thymines, and hypoxanthine.
It has recently been found that 3-methyladenine-DNA
glycosylase II will also remove natural bases from DNA,
especially purines, at significant rates. This finding has led to

the novel suggestion that the rate of excision of broadspecificity glycosylases is a function of the chemical bondstability in DNA. On this basis, damaged bases are excised
more readily than natural bases because their chemical bonds
are less stable [B57].
24. A specialized form of base-excision repair involves theremoval of mismatched DNA bases that occur as errors of
DNA replication or from the miscoding properties of damaged
bases. For example, 8-oxoguanine is a common product ofoxidative damage to guanine bases (paragraph 17); thisproduct is highly mutagenic because of its ability to miscodeANNEX F: DNA REPAIR AND MUTAGENESIS(polymerases incorporate adenine instead of cytosine opposite
8-oxoguanine, thus changing the DNA sequence). Cells have
evolved three different methods to deal with the formation of
8-oxoguanine: one glycosylase can correct the mismatch by
removing 8-oxoguanine from DNA, while a different
glycosylase can remove the mismatched adenine after DNA
replication [A27, T48]. Excision and resynthesis of themissing base occur as in the general base-excision repair
process. Additionally, another enzyme can remove theprecursor of 8-oxoguanine (8-hydroxy-GTP) from the nucleotide pool before it is incorporated into DNA [M51]. A numberof other mismatches, arising most commonlyin DNA replication and recombination, are corrected by a separate “longpatch” pathway, known as the MutHLS pathway, that issimilarly important in protecting cells from high frequencies
of mutation (see paragraph 165) [K38].
25. Where a damaged base is close to another damaged
base or to a single-strand break, which is the most simple
form of clustered damage, repair may be compromised.
Examination of model DNA substrates with base damage
on opposite strands, separated by different numbers ofbases, has shown that glycosylases do not repair both sitesof damage when they are very close to one another (1-3bases) [C56, C57, H49]. Further, attempted repair of bothsites of base damage can result in a DNA double-strand
break, because the DNA backbone is cut as part of therepair process.
26. In contrast to base-excision repair, nucleotide-excision
repair removes a whole section of single-stranded DNA
containing a site of damage, generally a bulky DNA adductcausing distortion of the double helix. These enzymes have toperform such functions as recognition of damage, cutting of
the strand at a specified distance either side of the damage,and unwinding and removal of the strand (Figure Ib). As
might be expected, at least 11 enzymes have already been
identified as components of nucleotide-excision repair [W52],
not including polymerase and ligase. The enzymes ofnucleotide-excision repair are highlyconserved from microbes
to humans, and this feature has been used to assist in theisolation of the genes encoding these functions. Several ofthese genes have been found to be mutated in humans, giving
rise to a series of disorders, including xeroderma pigmentosum and Cockayne's syndrome [H3]. Individuals inheriting
mutated nucleotide-excision repair genes are generally
sensitive to sunlight and chemical agents causing bulky
damage in DNA, but a few individuals show cross-sensitivity
to ionizing radiation [A2, R15]. Additionally, a small fractionof damage induced by gamma rays is not repaired in cells
derivedfrom individuals with xeroderma pigmentosum, whichsuggests that ionizing radiation induces some bulky damage(e.g. purine dimers) that cannot be removed by the baseexcision repair pathway [S56]. Alternatively, a fraction ofnon-bulky base damage (8-oxoguanine, thymine glycol) may
be removed by the nucleotide-excision repair system, particularly in long-lived cells such as neurons that sustain a great
deal of endogenous oxidative damage [R22]. These possibilities may explain why severe cases of xeroderma pigmentosum also suffer progressive neurological degeneration.

7

27. A surprising recent discoverywas that some nucleotideexcision repair enzymes are also involved in the normal
process of gene expression (transcription). Thus, when genes
are actively expressing, they require some of the samefunctions needed for repair, such as unwinding the DNA
helix, and it seems that the same proteins are used. Thisfinding explains the previously puzzling observation of a link
between human disorders with sensitivity to sunlight andthose with complex defects in gene expression (such astrichothiodystrophy [L70]).
28. Another important discovery about nucleotide-excision
repair is that it operates at different rates in different parts ofthe genome [H1]. Thus, activelyexpressing genes are repaired
much faster than the remainder of the genome. Much of thedetail of this process has now been elucidated: it is thoughtthat when damage occurs in a gene that is actively expressing,
the proteins involved in this process (the RNA polymerase IItranscription complex) stop working, and the stalled complex
acts as a signal to the repair proteins to go to the damage site.
This two-tier repair system has been found in organisms frombacteria to humans, and the protein mediating the signal tobring repair to the damage site has been identified in bacteria[S2]. The presence in the cell of a fast transcription-coupled
repair process has also been found to have genetic consequences: only one of the DNA strands of the duplex istranscribed, and only this strand is repaired rapidly. It has
been found in normal cells that most of the mutations induced
by DNA damage are in the non-transcribed strand, presumably because lack of fast repair allows the damage to interact
with other processes, causing mutations [M2]. In contrast,
repair-defective cell lines show a completely altered mutation
spectrum, with most mutations recovered in the transcribedstrand. Much of this detail has been established using UVlight damage, but the repair of other types of DNA damage,including certain forms of base damage induced by ionizing
radiation such as thymine glycols, is influenced by transcription-coupled processes [H1, C41]. Again, certain human sunsensitive disorders have been found to lack either the fast
repair path (Cockayne's syndrome) or the slower overall pathof nucleotide-excision repair (xeroderma pigmentosum group
C). Loss of the fast or slower pathways may also affect theclinical outcome of sun-sensitive disorders: Cockayne's syndrome does not result in cancer-proneness, while xeroderma
pigmentosum patients are highly prone to skin cancers [M2].
29. More severe forms of damage require yet moreresources for their correct repair. This is especially true ofdamage affecting both DNA strands simultaneously, since
there is no undamaged strand to act as a template forrepair. Severe damage may occur directly by a damaging
agent causing complex DNA changes or may arise during
the replication of unrepaired single-stranded DNA damage.
It is likely that such severe damage will be repaired byrecombination enzymes, which rejoin or replace damaged
sequences through a variety of mechanisms. In general,
there are two main types of recombination repair processes:
homologous recombination and illegitimate recombination,although site-specific recombination processes also occur.
The principles of recombination repair have been well

8

ANNEX F: DNA REPAIR AND MUTAGENESIS

established in micro-organisms, and it has recently beenfound that similar processes occur in human cells.
30. Homologous recombination takes advantage of thesequence identity between certain regions of DNA to repair
damage; such regions exist, for example, in the maternal andpaternal copies of chromosomes and in the duplicatedchromosome (sister chromatids) following DNA replication.
The DNA sequence, from which information is derived to
repair the damaged copy, must be identical over aconsiderable length ($200 base pairs). It is known in thebudding yeast (Saccharomyces cerevisiae), for example, that
homologous recombination is the main method for DNA
double-strand break repair. Several of the rad52 group of yeast
mutants were isolated on the basis of their extreme sensitivity
to ionizing radiation and have been shown to be defective inboth DNA double-strand break repair and homologous
recombination [G1]. In recombination, the broken 3' end of a
DNA strandinvadesan unbroken double-stranded homologue,
and resynthesis on this template re-forms the damaged strand(Figure Id). Separation of the joint product of this reaction
requires the activity of enzymes cutting and rejoining the
newly-synthesized DNA strands. Depending on which strands
are cut and rejoined, this reaction may also result in crossing
over (genetic exchange) of DNA strands.
31. Illegitimate recombination (including DNA endjoining processes) is a common mechanism for rejoining
broken DNA sequences in mammalian cells (Figure Ic).
When foreign DNA molecules are integrated into the
genome [R1] or when the genomic breakpoints of deletions
and rearrangements are analysed (Section IV.C), it isfound that these genomic sites show little sequencehomology. There appears to be more than one repair
pathway involved, and terms such as non-homologous end
joining and direct-repeat end joining are used to describe
different pathways in this Annex (see Section II.B.1). It
can be argued that illegitimate recombination is amechanism for rapidlyrejoining broken DNA ends without
the need for the complex machinery of homologous
recombination [R1]. It is also likely that because of thelarge amount of repetitive DNA sequence in mammaliancells, if the processes of homologous recombination weregenerally available in cells, there would be an intolerable
level of reshuffling of the genome. While homologous
recombination is thought to be a mechanism for repairing
DNA with little error, illegitimate recombination is likely
to cause alteration and/or loss of DNA sequence.
32. It is likely that both homologous and illegitimate
recombination processes are able to repair severe damage
in the genome. However, a surprising recent discovery inmammalian cells is that the some of the enzymes involved
in repairing radiation-induced breakage of DNA also takepart in a site-specific recombination process, V(D)J
immune-system recombination. This process assembles
functional immune genes from separate genomic regions,through somatic gene rearrangement, and is dealt with inmore detail in Section II.B.1.

33. There is also evidence that cells have specificsurveillance mechanisms for DNA damage and that thesemechanisms interface with other aspects of cellular metabolism such as cell-cycle progression [M3]. Thus it isenvisaged that when the genome, and perhaps other partsof the cell, sustains damage, a response mechanism is set
up to maximize the chance of repairing the damage (or insome cases to commit the cell to a programmed death). The
details of these mechanisms, as well as how the overall
response is coordinated, are not yet clear.
34. More than 50 genes are already known to affect the
repair of DNA damage in lower eukaryotic organisms suchas yeasts [F1], but this figure includes genes involved inprocesses such as cell-cycle checkpoints (Section II.B.2).
Additionally, new genes are being found continually, bothin searches for homologues of existing repair genes and in
genome mapping projects. In view of the numbers alreadydiscovered, the multiplicity of types of damage requiring
repair, and the recent discoveries of complexity in repair
pathways, it would not be surprising if the overall number
in humans is a few hundred genes. Therefore, a significant
fraction of the genome (paragraph 7) is devoted tomaintaining the integrity of DNA. Since the damage to
DNA from ionizing radiation is also very diverse, many ofthese genes will play a role in its repair.

C. SUMMARY
35. Ionizing radiation interacts with DNA to give manydifferent types of damage. Radiation track structure considerations indicate that the complexity of the damage increases
with linear energy transfer, and that this complexity may
distinguish radiation damage from alterations occurring
spontaneouslyand byother agents. Attempts to measure endogenous levels of damage have sufferred from high levels ofartefacts, and despite improved methods there are still large
margins of error in these estimates. At present, therefore, it isdifficult to compare radiation-induced levels of damage withthose occurring spontaneously, especially when damage complexity is taken into account. The importance of the relationship between spontaneous and induced levels of damage in thedetermination of low dose responses is taken up in Annex G,“Biological effects at low radiation doses”.
36. A large number of genes have evolved in allorganisms to repair DNA damage; the repair gene products
operate in a co-ordinated fashion to form repair pathways
that control restitution of specific types of damage. Repair
pathways are further co-ordinated with other metabolic
processes, such as cell cycle control, to optimize theprospects of successful repair.
37. It is likely that the simpler forms of DNA damage(single sites of base damage, single-strand breaks) arising
endogenously and from exposure to ionizing radiation will
be repaired rapidly and efficiently by base-excision repair
processes, so these types of damage are not normally aANNEX F: DNA REPAIR AND MUTAGENESIS

serious challenge to biological organisms. However,
because of the relatively large amount of base damage andsingle-strand breaks induced (Table 1), if base-excision
repair systems are compromised, the consequences would
be very serious for the cell and the individual. DNA
damage such as double-strand breaks represents a moredifficult problem for cellular repair processes, but more

9than one recombination repair pathway has evolved to copewith this damage. Damage caused by large clusters ofionizations in or near DNA, giving more complex DNA
alterations, may represent a special case for which separate
repair pathways have to come together to effect repair, or
where there is a consequent loss or alteration of the DNA
sequence as a result of incorrect or inadequate repair.

II. REPAIR PROCESSES AND RADIOSENSITIVITY

1. The identification of radiosensitive
cell lines and disorders
38. Individuals varyin their sensitivitytoionizing radiation.
Highly radiosensitive individuals have been detected when
they present for cancer therapy; these are seen as rare patients
suffering severe normal tissue damage after standard therapy
treatments. It has been possible to group some radiosensitive
patients into defined disorders, such as ataxia-telangiectasia
and the Nijmegen breakage syndrome, but others appear to be
asymptomatic (that is, with none of the symptoms of known
sensitivity disorders, but also discovered following treatment
for cancer). Additionally there are individuals who show lessextreme radiosensitivity, some of whom may be variants ofknown disorders such as ataxia-telangiectasia.
39. Ataxia-telangiectasia is the best described of radiosensitive disorders. It has a complex phenotype; cerebellar
ataxia, neuromuscular degeneration, dilated ocular bloodvessels (telangiectasia), immunodeficiency, chromosomal
instability, and a substantially increased incidence of somecancers and neoplasms are common to ataxia-telangiectasia
patients [B10]. Ataxia-telangiectasia is inherited primarily as
an autosomal recessive trait, although it has been suggested
that both radiosensitivity and cancer-proneness behave withsome dominance. The disease is progressive, with mostaffected individuals surviving only to adolescence or early
adulthood. Lymphocytic leukaemia and non-Hodgkin's
lymphoma appear to be the commonest forms of cancer, but
solid tumours in various organs are also associated withataxia-telangiectasia [H9]. Estimates of the frequency of thedisorder vary but suggest an average of about 1 per 100,000[P8, S12, W10].
40. The radiosensitive phenotype of ataxia-telangiectasia isalso readilydemonstrated in cells cultured from patients, using
cell survival and chromosome damage assays. For example ina survey comparing the survival of cells cultured from 42
normal individuals with those from 10 ataxia-telangiectasia
individuals following x-irradiation, the ataxia-telangiectasia
cells were, on average, 2.7 times more sensitive than thenormal cells (see Figure II) [C8]. Compared to normal cells,
an elevated frequency of chromosomal aberrations is foundboth spontaneously and after irradiation of ataxia-telangiec-

asia cells. Also, while irradiation of normal cells in the presynthesis (G0) phase of the cell cycle yields only chromosometype aberrations, both chromatid- and chromosome-type
aberrations are found in ataxia-telangiectasia [T1]. A striking
feature of ataxia-telangiectasia cells is their resistance toradiation-induced DNA synthesis delay: normal cells show arapid inhibition of DNA synthesis after irradiation, whileataxia-telangiectasia cellshave a delayed and/or much reduced
inhibition [P4].
1

SURVIVING FRACTION

A. RADIOSENSITIVITY IN MAMMALIANCELLS AND HUMANS

0.1

Normal individuals

Patients with ataxia-telangiectasia

0.01
0

1

2

3

DOSE (Gy)Figure II. Survival of human fibroblast cells after xirradiation as measured by their colony-forming ability[C8]. The range of D0 (the dose required to kill 63% of thecells) is 0.30.6 Gy in 10 patients with ataxia-telangiectasia
and 11.6 Gy in 42 normal individuals.

41. Nijmegen breakage syndrome is a clinically separate
radiosensitive disorder characterized by variable immune
deficiencies, microcephaly, developmental delay, chromoomal instability, and cancer susceptibility [B4, S7, V7, W4].
Lymphoreticular cancers again seem to characterize thisdisorder [S8]. Nijmegen breakage syndrome patients show no
ataxia or telangiectasia, but their cellular phenotype is very
similar to that of ataxia-telangiectasia [A17, J1, N6, T2].
Other patients with similarities to ataxia-telangiectasia andNijmegen breakage syndrome have been found and in somecases classified separately by genetic analysis ([C12, W5]; seealso below). One case has been reported of combined ataxiatelangiectasia and Nijmegen breakage syndrome and called ATFRESNO [C18]. Also, there are a number of reports of families
or individuals who show symptoms that partially overlap

10

ANNEX F: DNA REPAIR AND MUTAGENESIS

ataxia-telangiectasia and Nijmegen breakage syndrome; an
example would be a family with individuals showing either
ataxia-telangiectasia or a disorder involving ataxia, microephaly, and congenital cataract [Z3].
42. To add to this complexity, a number of individuals witha variant form of ataxia-telangiectasia have been described;
these patients may show a slower onset of symptoms and haveintermediate levels of cellular radiosensitivity [C4, C7, F2, J2,
T3, Y1, Z1].

DNA cross-linking agents (e.g. mitomycin C, diepoxybutane).
Additionally, a high proportion of deletions has been reported
in certain genes of Fanconi s anaemia cell lines, giving ahigher frequency of mutation in assays measuring loss ofheterozygosity (see paragraph 172) [S68]. There has beensome dispute over the extent to which Fanconi s anaemia cells
are sensitive to ionizing radiation; a lack of geneticclassification in these experiments may account for some ofthe variability found (see paragraph 68). However, when they
compared published data on the sensitivity of humanfibroblasts, Deschavanne et al. [D7] concluded that Fanconi s
anaemia was one of the few disorders for which sensitivity toradiation could be distinguished from that of normal cells.
777

43. In addition to individuals having multiple symptoms
associated with radiosensitivity, some otherwise normal
persons have been found to be highly radiosensitive. Woods
et al. [W9] described an apparently normal 13-year-old girlwhodeveloped multiple complications following radiotherapy
for Hodgkin's disease. Cells derived from a skin biopsy
showed a highly sensitive radiation survival response, similar
to that for ataxia-telangiectasia cells. Plowman et al. [P7]reported similar findings for a 14-year-old boy with acutelymphoblastic leukaemia; again, no ataxia-telangiectasia or
Nijmegen breakage syndrome-like symptoms were present,
but both whole-body and cellular radiosensitivity were as
extreme as for ataxia-telangiectasia. This individual has now
provided the first example of radiosensitivityin humans wherea defined cellular repair defect (in the ligase IV enzyme) canbe readily demonstrated; repair of radiation-induced DNA
double-strand breaks and interphase chromosome damage aresimilarly impaired (see also paragraph 66) [B15].
44. There is some evidence for ionizing radiation sensitivity
in several other cancer-prone disorders, although the
published data do not always agree on the degree of sensitivity(see also Section III.A). Bloom s syndrome is a rare autosomal
recessive disorder showing severe growth retardation, variable
immune deficiencies, and abnormal spermatogenesis [G20].
The age of onset of cancer is considerably earlier than normal;
about one third of surviving cancer patients with Bloom s
syndrome develop multiple primary tumours with no conistent pattern of cancer type or location. Individuals with
Bloom s syndrome develop a distinct facial rash from sunlightsensitivity, and their cells are not only hypersensitive toseveral different DNA-damaging agents but also show DNA
replica ion abnormalities [L47]. Genetic instability is seen inhigh levels of spontaneously-occurring chromosomal aberraions and sister chromatid exchanges; chromosomal sensitivity
to x rays has been found especially in cells in the G2 phase of
growth [A16, K30]. One case of Bloom s syndrome developed
oesophageal stricturefollowingstandard radiotherapytreat-ent
for lung cancer. This is very rarely seen in such treatment andis suggestive of hypersensitivity to radiation [K31].
7

7

7

7

45. Fanconi s anaemia is a cancer-prone disorder, mostcommonly presenting with acute myeloid leukaemia (15,000fold increased risk), although solid tumours are also found.
Bone marrow failure is a common diagnostic feature of thisdisorder, although symptoms may include congenital
malformations, abnormal skin pigmentation, and skeletal andrenal abnormalities [J15]. Fanconi s anaemia cells show highlevels of chromosomal aberrations and are hypersensitive to
7

7

46. The genes controlling radiosensitivity in humans will
not be identified simply by analysing radiosensitive disorders.
This is because mutations in many genes are deleterious to theextent that the development of a viable organism is inhibited.
This point was illustrated by the creation of a “knockout”
mouse for the UV-damage repair gene ERCC1 (knockout
meaning that both copies of the gene are inactivated). No
human variant for the ERCC1 gene has been found, and theknockout mouse dies before weaning, apparently as the resultof a massive load of (unrepaired) damage [M7]. Therefore, toexamine the full range of genes involved, it has beennecessary to derive radiosensitive mutant lines from cells in
culture. To this end, more than 50 mutant cell lines sensitive
to various genotoxic agents have been identified; many ofthese show some degree of x-ray sensitivity and are being used
to dissect repair pathways in mammalian cells [C16, H11].
These cell lines are especially useful for gene cloning, since
this has often proved difficult to achieve using cells derived
from human patients. It is possible at the present stage ofknowledge to group these mutant cell lines into several
categories based on their responses, and recently several of thegenes involved have been mapped or cloned (Table 2). As
radiosensitive cell lines have been developed in laboratories
around the world, almost all have been found to represent
defects in different genes. For this reason, and because
ionizing radiation produces a diversity of DNA damage, it isanticipated that a large number of the human genes involved
in determining radiation resistance remain undiscovered.
47. The discovery and analysis of the genetic basis ofradiation sensitivity is also being pursued through otherstrategies, including the biochemical analysis of repair
reactions and the purification of repair proteins, as well as
the identification of human repair genes by homology totheir counterparts in lower organisms (Section II.A.3). In
many instances, the genes discovered by these routes arefound to give rise to a high level of sensitivity whenmutated but to affect only a small fraction of the humanpopulation or to be inconsistent with life. However, some
genes affecting radiation sensitivity will probably havemore subtle effects, either because the particular genemutation only partially reduces gene product activity or
because the gene is not vital for cellular response toradiation. Studies exploring the latter types of response,
which may affect a much larger fraction of the humanpopulation, are described in Section III.A.

ANNEX F: DNA REPAIR AND MUTAGENESIS

11

Table 2
Classification of radiosensitive disorders and cell lines
Disorder /defective cell line(s)Human genedesignation a

Human
gene
location

Animal model
phenotype b

Probable DNA break repair defect and lossof cell-cyle control following damage

Ataxia telangiectasia
Nijmegen breakage syndrome
irs 2/V-series

ATM
NBS1
XRCC8

11q23
8q21
?

AT-like c

DNA double-strand break repair defective
and V(D)J recombination defectivexrs
XR-1/M10
V3/scid/SX9
180BR

XRCC5
XRCC4
XRCC7
LIG4

2q35
5q13
8p11q11
13q33q34

Immune deficiency dEmbryonic lethal
Immune deficiency dEmbryonic lethal

Sensitivity to many different agents;
some have DNA single-strand breakrepair defect and/or replication defect

46BR
Bloom’s syndrome
EM9
irs1
irs1SF
UV40

L1G1
BLM
XRCC1
XRCC2
XRCC3
XRCC9(FANCG)

19q13
15q26
19q13
7q36
14q32
9p13

Viable (acute anaemia)
Embryonic lethal
Embryonic lethal
Embryonic lethal








ATR
hRAD50hRAD51hRAD52
hRAD54hMRE11

3q2223
5q233115q
12p13.3
1p32
11q21

Embryonic lethal
Embryonic lethal
Embryonic lethal
ViableViableEmbryonic lethal

Type of defectRadiosensitivity inferred from structural
homology of genes to those known to be
involved in response to radiation damage
in lower organisms

a
b
c
d







XRCC = x-ray cross complementing gene.
Knockout mice except for XRCC7; () indicates no model yet available
Symptoms similar to ataxia telangiectasia (see paragraph 62).
Severe combined immune deficiency.

2. Mechanisms of enhanced sensitivity
in human disorders
48. In addition to being radiosensitive, ataxia-telangiectasia
cell lines show enhanced sensitivity to agents that have incommon an ability to damage DNA molecules by producing
highly reactive chemical radicals, causing both base damage
and sugar damage, leading to breakage of DNA strands.
Ataxia-telangiectasia cells have been found to be hyperensitive to a variety of chemicals that cause such DNA
damage through radical action (bleomycin, neocarzinostatin,hydrogen peroxide, streptonigrin, phorbol ester [M5]). Also,
inhibitors of DNA topoisomerases that can trap these enzymes
during DNA-strand passage, leaving open breaks, are more
effective at inducing chromosomal damage and cell killing in
ataxia-telangiectasia cells than in normal cells [C1, H7, S11].
More recently, ataxia-telangiectasia cell lines have been foundto be hypersensitive to restriction endonucleases; these
enzymes produce only double-strand breaks in DNA by direct
enzymatic cutting [C17, L56]. There is also evidence ofmodest chromosomal hypersensitivity in some ataxiatelangiectasia lines to agents such as UV light, especiallywhen irradiated in extended G1 phase, possibly because of theexcessive production of breaks when DNA synthesis isattempted [E1, K4].
49. Experiments varying the time component either during
or following irradiation have revealed the general nature of
the defect in ataxia-telangiectasia cells. It was shown that
normal cells held after irradiation in a non-growing state hadsome recovery (or sparing) from lethal effects, while ataxiatelangiectasia cell lines showed little or no sparing [C9, U15,
W2]. More strikingly, irradiation at low dose rates, where thesame dose was given over a period of days instead of minutes(factor of 500 difference in dose rates) showed a very large
sparing effect on normal cells and little or no effect on ataxiatelangiectasia cells [C10]. These observations are consistent
with an inability of ataxia-telangiectasia cells to recover fromradiation damage, and they also show that the defect cannot
be abrogated simply by allowing more time for damage
restitution.
50. Lack of a sparing effect appears to be typical of aparticular class of radiosensitive cell lines. Thus lines that are
known to have a defect in the repair of DNA double-strand
breaks, for example the xrs series and XR-1, also lackrecovery under irradiation conditions in which their normal
counterparts show a large sparing effect [S14, T7]. Similarly,
yeast radiosensitive lines that are unable to rejoin doublestrand breaks, because of a defect in recombination (rad50,
rad51, rad52), also lack sparing [R2, R3]. A substantial bodyof data in yeast supports the contention that the double-strand
break is the DNA damage most likely to be lethal to cells, andthat its repair is responsible for the recovery seen undersparing conditions [F3].
51. In contrast to these cellular studies implicating strand
breakage as the type of DNA damage involved in the ataxiatelangiectasia defect, it has been difficult to prove that ataxiatelangiectasia cells have a break-repair defect at the molecular

12

ANNEX F: DNA REPAIR AND MUTAGENESIS

level [M5]. Recently, irradiation at 37EC with low-dose-rate
gamma rays has shown a small increase in DNA doublestrand breaks following repair ("residual damage") in ataxiatelangiectasia cells relative to normal cells [B9, F10, F16].
However, cytogenetic studies have provided more satisfactoryevidence for a break-repair defect: a significantly elevatedfraction of unrestituted chromosomal breaks remain in ataxiatelangiectasia cells after irradiation [C11, T1]. Support forthese findings has been obtained from the measurement ofboth DNA double-strand breaks and chromosomal breaks(using prematurely condensed chromosomes to allow rapid
analysis) after gamma irradiation of normal and ataxiatelangiectasia lymphoblastoid cells at different phases of thecell cycle [P5]. A consistent decrease in the rapid componentof repair was found in ataxia-telangiectasia relative to normal
cells; this decrease was small and usually statistically nonsignificant for DNA double-strand breaks, but larger andsignificant for chromosomal breaks. Differences in amountsof residual chromosomal damage between normal and ataxiatelangiectasia cells give a close, but not exact, approximation
to their relative survival levels after irradiation [C14, P5].
52. Hamster cell lines showing strong similarities to
ataxia-telangiectasia have been isolated and their responses
characterized (the irs2 line [J5] and the V-series [Z5]).
These lines are hypersensitive to agents known to cause
DNA breakage, have radioresistant DNA synthesis, andhave no measurable biochemical defect in DNA breakrepair [T8, Z6]. It has also been shown that the radiation
sparing effect is absent in irs2, while it is present in otherlines that have similar radiosensitivity but do not show
ataxia-telangiectasia-like characteristics (such as irs1 andirs3) [T30].
53. Overall, these studies strongly support the view thatthe increased sensitivityof ataxia-telangiectasia and related
cell lines to agents such as ionizing radiation derives froman inability to recover from DNA breakage, leading to ahigher level of residual chromosomal damage. However,
the molecular mechanisms leading to radiosensitivity inthis disorder are still not fully understood, despite
considerable recent progress in defining the function of theataxia-telangiectasia gene product (see Section II.A.3).
54. The functional defects in other cancer-prone
disorders have also not been well characterized. Primarycells from Fanconi s anaemia patients have spontaneous
delay and arrest in the G2 phase of growth, as well as anincreased frequency of chromosomal aberrations [J15] andrecombination [T39]. G2 delay and aberration frequency
increases are corrected by lowering the oxygen tension
during growth, leading to the suggestion that reduced
detoxification of oxygen radicals may be responsible for thephenotype [C62, J17]. However, immortalized Fanconi s
anaemia fibroblasts have lost this oxygen effect, showingthat this factor is not a basic (or underlying) defect [S48].
In Bloom s syndrome, the high frequency of sister
chromatid exchanges and specific types of chromosome
aberrations suggested a defect in DNA repair and/or DNA
replication.
77

7

3. Analysis of genes determining
radiosensitivity
55. The classification of radiosensitive disorders and celllines into genetic groups, followed by mapping and cloning of
the affected genes, has dramatically increased knowledge ofthe molecular mechanisms of recovery from DNA damage.
Once the affected gene has been cloned, its sequence may
reveal the nature of the gene product (protein), because ofsimilarities to known genes. Gene sequence data from at-risk
groups will also allow deleterious mutations to be identified,and permit analysis of the role of these genes in disorders suchas cancer. Manipulation and expression of the gene under
defined experimental conditions allow specific functions to bestudied in cells and in animals. Animal models of the humandisorder can be created by replacing the normal pair of genes
with defective copies (a knockout animal; see paragraph 46)
and assessing the resulting phenotype.
56. Genetic classification of the ataxia-telangiectasia disrder initially indicated that several different genetic groups
might exist [C3, J2], but mapping and cloning of a gene(ATM) found to be mutated in patients has cast doubt on thisdesignation. The genetic mapping data, based on ataxiatelangiectasia family studies, mostly placed the affected gene
into one chromosomal region, 11q23.1 [G2, Z2]. Positional
cloning procedures in this region led to the identification of
the ATM gene, which has homology to a gene family
encoding PI-3 kinases [B31, S24, S25]. The PI-3 kinase
family contains a number of large proteins involved in cellcycle checkpoints, the regulation of chromosome-end length,and DNA break repair, including site-specific recombination(see Section II.B.1). It is therefore likely that ATM and othermembers of this family are involved in the detection of certain
types of DNA alterations and may coordinate response bysignalling these changes to other regulatory molecules in thecell [J8, K3, S25, T29].
57. Analysis of mutations in the ATM gene of ataxiatelangiectasia patients showed that the majorityare compound
heterozygotes (i.e. the mutations in the two gene copies derivefrom independent events) and that these commonly lead to an
inactive, truncated protein [G19, M30]. However, individuals
from 10 families in the United Kingdom with less severe
symptoms (paragraph 42) all have the same mutation in one
copy of ATM (a 137-bp insertion caused by a point mutation
in a splice site) but differ in the mutation in the other gene
copy. The less severe phenotype appears to arise from a low
level of production of normal protein from the insertioncontaining gene copy. Two more families with this less severe
phenotype have mutations leading to the production of analtered but full-length protein, again suggesting that theseverity of the symptoms in ataxia-telangiectasia is linked to
the genotype of the individual [L41, M30]. However, it ispossible that individuals with less severe symptoms have an
increased risk of developing specific types of cancer [S78] (seealso paragraph 139).
58. A further two families presenting with many of thesymptoms of ataxia-telangiectasia (paragraph 39) but

ANNEX F: DNA REPAIR AND MUTAGENESIS

without dilated ocular blood vessels failed to show
mutations in the ATM gene. The cellular characteristics offamily members, such as radiation sensitivity and DNAsynthesis delay (paragraph 40), were mostly intermediate
between those of classical ataxia-telangiectasia patients andnormal individuals. Examination of other genes implicatedin the repair of DNA double-strand breaks revealed that thehuman homologue of the yeast MRE11 gene (paragraph74) was mutated in these families [S82].
59. A rare form of leukaemia, sporadic T-cell prolymphocytic leukaemia (T-PLL), shows a high frequency of ATM
mutations; although some of the changes identified in the
ATM gene were rearrangements, most mutations in T-PLL
were single DNA base-pair changes in the kinase region of
the ATM gene and did not lead to protein truncation [V5, Y8].
This finding has prompted the suggestion that the ATM geneacts as a tumour suppressor in cells that may develop T-PLL.
There is no evidence for an involvement of the ATM gene in
T-cell lymphoblastic leukaemias [L58], but in B-cell chronic
lymphocytic leukaemia (B-CLL), 34%-40% of tumour
samples showed about 50% reduction in levels of ATM
protein [S70, S76]. ATM mutations were detected in thetumours of 6 out of 32 patients (18%); also 2 of these 6
patients had mutations in both tumour and normal cell DNA,
indicating that they were carriers of an ATM gene defect (i.e,they inherited a mutation in one copy of the ATM gene; seeparagraph 137 et seq.) [S76]. Similar results were obtained ina separate study [B62], and although they are based at present
on small samples, the data suggest that the frequency of ATM
mutations in the normal cells of patients developing B-CLL
may be much higher than in the general population [B62,
S76]. Patients with ATM deficiency also had significantly
shorter survival times [S70].
60. Study of the ATM protein has shown that it has a
nuclear location and is expressed in many different humantissues. Additionally, ATM protein does not increase inamount in response to cell irradiation, consistent with the ideathat it is part of a DNA-damage-detection system rather thanbeing regulated in response to DNA damage [L41]. However,the ATM protein does associate with DNA, and thisinteraction increases when the DNA is irradiated [S77]. The
ATM gene has a complex structure with multiple transcription
start sites, leading to messenger RNAs of different length andpredicted secondary structure. This multiplicity of ATM
transcripts may allow cells to modulate ATM protein levels in
response to alterations in environmental signals or cellular
metabolism [S40].
61. Initially it was also suggested that the Nijmegen
breakage syndrome involves more than one genetic group[J1], but recent evidence shows that only one gene is involved[M41] and that this gene maps to chromosome 8q21 [M41,
S59]. These data support clinical findings (paragraph 41)
showing that the Nijmegen breakage syndrome is a separate
radiosensitivedisorder, distinct from ataxia-telangiectasia. The
cloning of the gene (NBS1) mutated in this syndrome has
confirmed this (paragraph 74), with the majority of patients
carrying small deletions in this gene [M45, V6].

13

62. Knockout mice that lack a functional homologue of theataxia-telangiectasia gene (Atm) have recentlybeen bred; theyshow many of the symptoms of the human disorder, but also
give further insights into the action of this gene [B33, E9,
X3]. For example, the Atm knockout mice are viable but
growth-retarded and infertile. They are also very sensitive toacute gamma radiation; at a whole-body dose of 4 Gy, about
two thirds of the Atm knockout mice died after 5-7 days, while
normal and heterozygous mice remained without morbidity
after two months [B33]. Primary cells derived from the Atm
knockout mice also show many of the characteristic featuresof cells from ataxia-telangiectasia patients [X4]. The cells
grow poorly, are hypersensitive to gamma radiation, and failto undergo arrest of the cell cycle following irradiation (seealso paragraph 102). The Atm gene product was found tolocate tohomologous chromosomes as theyassociate (synapse)
at meiosis in germ cells [K27]; loss of fertility in the knockout
mice results from failure of meiosis due to abnormal synapsis
and subsequent chromosome fragmentation [X3]. Immune
defects occur in these mice, and the majority develop thymic
lymphomas and die before four months. Based on theunderstanding to date of the defective response to DNA
breakage in ataxia-telangiectasia, the meiotic failure andimmune defects in these mice could both relate to an inability
to respond to “programmed” DNA double-strand breaks (sitespecific breaks that occur in the course of normal cellular
processes). Such breaks are thought to be essential in meiosis,
to drive the process of meiotic recombination, and they are
required for V(D)J recombination in immune systemdevelopment (see paragraph 82).
63. Unlike in the human disorder, no evidence was foundof gross cerebellar degeneration in Atm knockout mice
aged 1!4 months, but it is possible that the animals aredying too early for this symptom to be revealed [B33, X4].
However, behavioural tests indicated some impairment of
cerebellar function [B33], and detailed studies of the brain
in these animals showed that Atm deficiency can severely
affect dopaminergic neurons in the central nervous system[E14]. There is an almost complete absence of radiationinduced apoptotic cell death (Section II.B.3) in thedeveloping central nervous system of Atm-deficient mice,
while the thymus shows normal levels of apoptosis after
irradiation [H48]. Additionally, elevated levels of oxidative
damage were recorded in mouse tissues affected by the lossof ATM, especially the cerebellum [B63].
64. In yeast cells, the closest sequence homologue to the
ATM gene product is the Tel1 protein; mutations in the TEL1
gene are associated with shortened chromosome ends
(telomeres) and genetic instability (three- to fourfold
increased levels of mitotic recombination and chromosomeloss) [G22]. It has been known for many years thatchromosomes in ataxia-telangiectasia cells show a relatively
high incidence of telomere fusions, and recent studies haveshown that preleukaemic cells from ataxia-telangiectasia
patients have an increased rate of telomere loss [M29]. This
loss may contribute to chromosomal instability [R40].
However, as yet the relationship between the telomere fusions
and tumorigenesis in ataxia-telangiectasia is not clear.

14

ANNEX F: DNA REPAIR AND MUTAGENESISInterestingly, loss of the TEL1 gene does not lead to x- or
gamma-ray sensitivity [G22, M31], but the combined loss of
TEL1 and another gene in the same family (ESR1/MEC1; seenext paragraph) gives extreme sensitivity to gamma rays,suggesting that these two genes may functionally overlap inprotecting the cell against radiation damage [M31]. In fission
yeast, following prolonged growth, loss of these two genes led
to circular chromosomes lacking telomeric sequences [N26].
65. A further member of the human PI-3 kinase family,
related to ATM, has been found through homology searches[B32, C38]. The gene (named ATR or FRP1) encodes aprotein that is most closely similar to the fission yeast rad3
gene product, which is itself structurally and functionallyrelated to the budding yeast ESR1/MEC1 and the fruit fly
mei-41 gene products. Mutations in these genes render theyeast or flies sensitive to killing by both ionizing and UV
radiation, and are known to play important roles in mitotic
and meiotic cell-cycle controls. Expression of an inactive
form of ATR at a high level in human fibroblasts increased
radiation sensitivity by a factor of 2-3 and abrogatedradiation-induced G2 arrest (see paragraph 108). Additionally, cells with defective ATR develop abnormal
nuclear morphologies, which may indicate further cellcycle perturbations [C48, W50]. The ATR gene product isexpressed at a high level in germinal tissue and localizes
along unsynapsed meiotic chromosomes [K27], thereby
playing a role that is complementary to that of the ATM
gene product (paragraph 62). Thus in addition to theirroles in mitotic cells, ATM and ATR gene products may be
involved in the co-ordination of meiotic chromosome
synapsis, perhaps by signalling breaks and monitoring
repair synthesis to guard against genetic instability.
66. Some other genes affecting radiosensitivity inhumans have also been mapped and cloned. Following thecloning of the human DNA ligase I gene, it was discoveredthat this was defective in a unique individual with growth
retardation, immunological abnormalities, and cellularsensitivity to a variety of DNA-damaging agents, including
radiation [B5]. The genes for other human DNA ligases
have also been cloned using homology searches or protein
purification [C31, W19], and subsequently another uniqueradiosensitive individual with impairment of double-strand
break rejoining (described in paragraph 43) was found to
be defective in DNA ligase IV [R38]. It has been
established in yeast and mammalian cells that DNA ligase
IV is specifically involved in the repair of DNA breaks by
non-homologous end joining (Section II.B.1) [S64, T40,
W46]. DNA ligase III has been implicated in the baseexcision repair pathway (see paragraph 78).
67. Bloom s syndrome patients of different ethnic origins(Ashkenasi Jewish, French-Canadian, Mennonite, andJapanese) were found by cell fusion analysis to fall into one
genetic group [W36]. The gene defective in Bloom s
syndrome (named BLM) has been positionally cloned andshown to be similar to the bacterial RecQ helicase, a typeof enzyme that opens up the DNA helix and is associatedwith genetic recombination in bacteria [E6, K39].
77Knocking out the gene homologous to BLM in mice resultsin embryonic growth retardation and lethality, apparently
because of a wave of increased apoptotic death [C61]. The
yeast homologue of the BLM gene product interacts with
DNA topoisomerase enzymes, known to be required for the
resolution of interlocking DNA molecules following
replication. In the absence of the BLM gene product, it issuggested that replicated DNA (in mitosis or meiosis) is
entangled and may give rise to sister-chromatid exchange
and chromosomal non-disjunction [W35]. Support for thisidea comes from studies with the fission yeast, where therecQ helicase is required for the recovery from cell-cycle
arrest during DNA replication following DNA damage;absence of the recQ helicase leads to an increase in the rateof genetic recombination [S60].
68. Cells derived from Fanconi s anaemia patients havebeen classified into eight genetic groups [J16, J19, S46],
consistent with the heterogeneity of symptoms found(paragraph 45). To date, three of these genes have been
mapped, FANCA to chromosome 16q24 [G21, P20],FANCC to 9q22 [S46], and FANCD to 3p22-26 [W37].
The FANCA and FANCC genes have now been cloned[F9, L43, S47]; these gene sequences predict proteins thatare structurally different from each other and from otherknown proteins. Mutations of the FANCA gene wereanalysed in 97 patients from different ethnic groups with
Fanconi s anaemia; the majorityof mutations detected were
either DNA base-pair alterations or small deletions andinsertions scattered throughout the gene, with a smaller
number of large deletions [L49]. The FANCC gene productis a cytoplasmic protein [Y7], but it interacts with the
FANCA gene product, and the complex translocates to the
nucleus [K40]. While this nuclear localization is consistent
with a possible role in DNA repair, the precise function ofthese gene products remains unclear. FANCC also hasbinding sites for the p53 tumour suppressor protein(paragraph 100), and binding of p53 to the gene canregulate its expression [L46].
7

7

69. One of the more recently identified genetic groups of
Fanconi’s anaemia, FA-G, has been found to be caused bymutation of a previously-identified gene, XRCC9 [D18].
This gene was identified by the complementation of arodent cell line sensitive to a variety of DNA-damaging
agents including a twofold enhanced sensitivity to x rays,
and is potentially involved in post-replication repair ofDNA [L59] (Table 2).
70. Human genes implicated in the repair of radiationinduced DNA damage have also been cloned by theirstructural homology to genes involved in specific repair
pathways in lower organisms. For example, it is known
that the main pathway for the repair of DNA double-strand
breaks in lower organisms involves homologous recombination (paragraph 30); in budding yeast, the genes
responsible are RAD50, RAD51, RAD52, RAD53, RAD54,
RAD55, RAD57, MRE11, and XRS2 (often called theRAD52 group of genes). Mutations in these genes render
the yeast cells defective in mitotic and/or meiotic

ANNEX F: DNA REPAIR AND MUTAGENESIS

recombination and sensitive to ionizing radiation. At least
some of the products of these genes act together in a multiprotein complex to effect recombination [H27, J14, U18].
In yeast, functional relationships have also been foundbetween proteins of the PI-3 kinase family and some of theRad52 group proteins. For example, the activity of theRAD53 gene product, another protein kinase, is regulated
by both ESR1/MEC1 and TEL1 gene products, suggestingthat the Rad53 protein acts as a signal transducer in the
DNA damage response pathway downstream of theseATM-like proteins [S43]. Sequence similarities have led tothe cloning of human homologues of some of the yeast
RAD52 gene group: hRAD50 [D16], hRAD51 [S10, Y6],
hRAD52 [M10, S44], hRAD54 [K28], and hMRE11 [P22].
Preliminary evidence has been obtained from their abilities
to partially correct the defects in radiosensitive yeast celllines that some of these genes, or their mouse homologues,
are involved in the repair of radiation damage [K28, M37].
Further, knocking out the RAD54 gene in both the fruit fly[K41] and mouse [E12] confers radiation sensitivity and a
defect in homologous recombination. There is also
evidence for regulated expression of these recombination
proteins through the cell cycle, with levels of RAD51 andRAD52 increasing during the S phase and peaking in G2
in human and rodent cells [C5].

15

strand breaks can be partially corrected by introduction of
the normal gene, while mutant BRCA1genes fail to restore
these defects [S86]. A product of a second gene associatedwith familial breast cancer susceptibility, BRCA2, hassimilarly been shown to interact with RAD51 [M42, S61],
as well as with p53 [M46]. BRCA1 and BRCA2 are highly
expressed in rapidly proliferating cells, with expression
highest at the start of S phase of the cell cycle [R23].
Analysis of mutations in the BRCA genes has shown thatfamilies with a high proportion of breast cancer tend tohave mutations in different parts of the gene from families
having a predisposition to ovarian cancer [G37, G38].
Despite their importance in cancer predisposition, themolecular function of the BRCA genes is unknown, and thefinding of interaction with RAD51 provided the first cluethat their role may be in DNA repair. As noted inparagraph 62, the ATM protein also has a specific patternof localization to meiotic chromosomes, and loss of ATM
can disrupt the localization pattern of RAD51. Thus,
through their connections with RAD51, these data provide
for the first time a mechanistic link between four different
genes involved in cancer susceptibility (ATM, TP53,
BRCA1, and BRCA2).

71. The human RAD51 gene product has been shown tobind DNA and thereby underwind the double helix, anearly step in the recombination process [B34]. The RAD51
gene in yeast is not essential for cell survival, but recently
the homologous gene has been knocked out in mice andsurprisingly was found to give embryonic lethality [L42,
T34]. Knockout embryos arrested early in development but
progressed further if there was also a mutation in the geneencoding the tumour suppressor p53 (TP53), possibly becauseof a reduction in programmed cell death (see paragraph 112).
This may suggest that RAD51 in mammals has functions inaddition to recombination and repair, and in a separate study
the human RAD51 protein has been shown to directlyinteract
with p53 [S45]. Cells from the mouse Rad51 knockout were
hypersensitive to gamma rays, although this response has notbeen quantified, and show chromosome loss in mitotic cells[L42]. In mitotic cells, the mouse RAD51 protein is found toconcentrate in multiple foci within the nucleus at the DNAsynthesis (S) phase of the cell cycle [T37]. In meiotic cells,
RAD51 is found on synapsing chromosomes and disappears
shortly after this stage [P23], but this pattern of localization of
RAD51 is disrupted in Atm-deficient mice [B47].

73. Mice have been bred with a knockout of the Brca1 geneor the Brca2 gene; like Rad51 knockout mice, they were bothfound to arrest early in embryonic development [H40, L50,
S61]. However, viable mice have been bred with a mutationin Brca1 or Brca2 that does not completely inactivate thegene. A conditional deletion of the Brca1 gene in mice,
confining the defect to the mammary glands, gave a low
frequency of mammary tumour formation with long latency(see also paragraph 106) [X7]. Embryonic fibroblasts derived
from Brca1-deficient mice proliferate poorlyand show genetic
instability [X8] and a reduction in the frequency of homologous recombination [M50]. About one third of the Brca2deficient mice survived to adulthood but showed small size,
poor tissue differentiation, absence of germ cells, anddevelopment of thymic lymphomas [C59]. Cells derived fromviable Brca2-defective mice proliferate poorly, apparently asa result of spontaneously high levels of p53 and p21, causing
cell-cycle arrest (see paragraph 100) [C59, P32]. These cells
accumulate high levels of chromosomal aberrations and showenhanced sensitivity to DNA-damaging agents, including
x rays [M48, P32]. The evidence linking the BRCA genes to
DNA repair processes suggests that they may not function as
tumour suppressors but are involved in the maintenance ofgenome integrity.

72. The pattern of cellular localization found for the
RAD51 protein has also been found for the product of agene commonly mutated in familial breast and ovariancancer patients, BRCA1; subsequently, RAD51 andBRCA1 proteins were shown to interact directly [S51].
Brca1-deficient mouse cells show a modest increase insensitivity to ionizing radiation and have a reduced
capacity to repair base damage in transcribing DNA(transcription-coupled repair, paragraph 28) [G34]. In
human BRCA1-deficient cells, increased sensitivity to cellkilling by gamma rays and reduced repair of DNA double-

74. The yeast RAD50, MRE11, and XRS2 gene products
assemble into a multi-protein complex that is implicated in
the processing of broken DNA, as well as in a number ofother functions, including meiotic recombination and telomere
maintenance [H46]. This multiplicity of roles for the complex
may arise from its nuclease activities on both single- anddouble-stranded DNA ends that trim up the DNA in
preparation for end-joining and homologous recombination
processes. Human gene products with similar functions havebeen identified (paragraph 70); significantly, the gene mutatedin the Nijmegen breakage syndrome (NBS1) has been found16

ANNEX F: DNA REPAIR AND MUTAGENESIS

to be the functional homologue of XRS2 (paragraphs 41 and61) [C54, M45, V6], and individuals with a variant form of
ataxia-telangiectasia have mutations in the human homologueof MRE11 (paragraphs 42 and 58) [S82]. The NBS1 geneproduct contains motifs that are commonly present in proteins
involved in cell-cycle regulation and DNA-damage response,suggesting that like ATM, this gene has a role in thesignalling mechanism following damage to DNA by ionizing
radiation. Study of the purified protein shows that DNA
unwinding and nuclease activities of RAD50/MRE11 arepromoted bythe presence of NBS1 [P34]. The human RAD50
and MRE11 proteins co-localize in cell nuclei following
ionizing radiation damage (but not after UV irradiation); theirsites of localization were distinct from those of the RAD51
protein, consistent with their different roles in DNA repair[D17]. Co-localization of MRE11/RAD50 proteins was much
reduced in ataxia-telangiectasia cells, suggesting that ATM
gene signalling is important for the assembly of the breakrejoin complex [M28]. Additionally, the BRCA1 protein has
been shown to colocalize and interact with RAD50, andradiation-induced sites of localization including MRE11,
RAD50 and NBS1 proteins were much reduced in breastcancer cells lacking BRCA1 [Z12].
75. Human genes encoding enzymes responsible for the
repair of the numerous forms of base damage caused by
DNA-damaging agents, including ionizing radiation, arealso being discovered [L69, S49]. A number of key
enzymes (DNA glycosylases, endonucleases) in the baseexcision repair pathway have been isolated by biochemical
purification, followed by protein sequencing and gene
identification, or through their homologies to known
enzymes in lower organisms. For example, the hOGG1
glycosylase [R41] removes 8-oxoguanine opposite acytosine (paragraph 24), while the hNTH1 glycosylase[A28] removes oxidized pyrimidines such as thymine
glycol (paragraph 17). The pathway of base-excision repair
that results in the incorporation of a single nucleotide(“short-patch repair”) has been reconstituted under cellfree conditions, using purified enzymes [K24].
Additionally, a second “long-patch repair” pathway has
been identified, in which between two and six nucleotides
are replaced following repair of a reduced or oxidized baseless site; this pathway also requires the structure-specificnuclease DNase IV (also known as FEN1) to remove thedisplaced nucleotides during repair [K42]. The
reconstitution of base-excision repair of oxidized
pyrimidines in DNA has revealed that hNTH1 glycosylase
is strongly stimulated by one of the proteins involved innucleotide-excision repair, XPG (xeroderma pigmentosum
group G protein). The XPG protein binds to non-paired
regions of DNA, and acts as a structure-specific nuclease
where the unpaired region is greater than five base pairs,
as will happen at sites of bulky damage. However, at sitesof oxidative base damage it seems that the unpaired regionis less than five base pairs, and instead of cutting DNA the
XPG protein promotes the activity of the hNTH1 inremoving the base damage [K46]. These findings may
explain the extreme symptoms of some patients lacking
XPG, including growth failure and neurologicaldysfunction, and the early death of mice carrying aknockout of the Xpg gene [H51] (while mice that are
totally defective for nucleotide-excision repair such as
those defective in Xpa are viable [N27]).
76. The main endonuclease involved in the repair of baseless sites (Figure Ia) in human cells, known variously as
HAP1, APE, APEX, or Ref-1, was cloned in several
laboratories following biochemical purification and was foundto have a surprising additional function when compared withthe bacterial enzyme. It stimulates the DNA-binding activityof transcription factors involved in signal transduction such as
Fos, Jun, NFκB, and p53 by reduction/oxidation mechanisms[X5]. Signalling and repair responses to oxidative damage
may therefore be coordinated through this one enzyme; thismay be especially important during tissue proliferation, sinceRef-1 knockout mice die during embryonic development [X6].
Knockout mice have been produced for several other genes inthe base-excision pathways, and a number of these have also
proved to be embryonic-lethal, showing their importance forthe normal functioning of cells and tissues [W45]. However,a knockout mouse for the broad-specificity glycosylase 3methyladenine-DNA glycosylase II was recently shown to be
viable [E13], possiblybecause of its mode ofaction (paragraph23) and the likelihood that other more specialized glycosylases
can substitute for it in removing damaged bases.
77. The abundant enzyme poly(ADP-ribose) polymerase,
also known as PARP, is rapidly recruited to sites of DNA
breakage following irradiation, where PARP transiently
synthesizes long, branched chains of poly(ADP-ribose) on
itself and other cellular proteins. These chains aredegraded by another enzyme, poly(ADP-ribose)
glycohydrolase, with a half-life of only a few minutes. The
role of PARP in response to DNA damage, which has long
been in dispute [C49], has been clarified from recent
experiments in which the Parp gene was knocked out inmice [M9]. These mice are viable and fertile, although
adult size is smaller than average and litter sizes are
smaller than for normal mice. Following whole-body
irradiation (8 Gy gamma rays), the Parp knockout micedied much more rapidly than normal mice from acuteradiation toxicity to the small intestine; the survival halftime of these irradiated PARP-deficient mice wascomparable to that of irradiated Atm knockout mice(paragraph 62). PARP-deficient cells or cells in which amutated PARP is expressed [S63], show increases inchromosomal aberrations, sister-chromatid exchanges, andapoptosis following DNA damage. The average length oftelomeres is significantly shorter in PARP-deficient mouse
cells, leading to an increased frequency of chromosomal
fusions and other aberrations [D21]. Recent biochemical
data suggest that PARP has an important function as amolecular sensor of DNA breaks, especially single-strand
breaks, and its absence reduces the efficiency of baseexcision repair [O4]. In addition, it has been suggested thatthe synthesis of poly(ADP-ribose) chains causes negativecharge repulsion of damaged DNA strands, preventing
accidental recombination between homologous sequences[S62].

ANNEX F: DNA REPAIR AND MUTAGENESIS

78. To date, studies with radiosensitive mammalian lines
have identified more than eight genetic groups [J3, T9, Z13].
The genes responsible have provisionally been named the
XRCC (for X-Ray Cross-Complementing) group, despite thefact that some of the cell lines are not primarily sensitive tox rays. The human gene corresponding to the first group,
XRCC1, has been cloned and encodes a protein interactingwith DNA ligase III [C2]. Further, the XRCC1 protein has
been shown to associate with DNA polymerase β [C39, K29],
which fills in the gaps created during the repair of damaged
bases. It is suggested that XRCC1 may act as a scaffold
protein in the final steps of base-excision repair (Figure Ia),
supporting the activity of DNA polymerase β and DNA ligase
III [K29]. The XRCC2 gene [C66, L65, T35] and the XRCC3
gene [L65]have been cloned recently, and both genes havestructural homology to the yeast and human RAD51 genes[T47]; it is likely, therefore that they are involved in repair ofdamage by homologous recombination. Other human genes
recentlycloned using mammalian cell lines (XRCC4, XRCC5,
XRCC7) are involved in the repair of radiation-induced DNA
double-strand breaks as well as in immune generecombination, as detailed below (Section II.B.1). Current
mapping data place all of the remaining genes on different
chromosomes, and none maps to the location of characterizedradiosensitive human syndromes (Table 2).
4. Summary
79. Several radiosensitive human disorders and examples ofindividual radiosensitivity have been identified in recent
years. The sensitivity in these individuals is characterized bya greatly increased susceptibility to cancer, although thiscancer is not necessarily radiation-induced. Many radiosen-

17

sitive lines of cultured cells have also been established, andthese have been useful for identifying the genes involved andfor functional studies. In general, it has been found thatenhanced sensitivity arises from an inability to recover fromDNA damage, because of a reduction in damage detection
and/or repair processes. Reduction of repair capacity
commonly leads to a lack of low-dose-rate sparing and ahigher level of genetic changes.
80. Considerable progress has recently been made in
defining repair gene functions in human and othermammalian cells, and a summary of well-defined repair
pathways for damage by ionizing radiation is shown in Figure
III. A number of important genes have been cloned, including
the gene ATM, which determines sensitivity in the ataxiatelangiectasia disorder. Studies with the ATM gene productand the production of mice defective for the gene suggest thatit participates in the detection of DNA double-strand breaks
and passes this information on to other important molecules
regulating cellular response processes. Other recently
discovered genes act directly in the repair of radiation
damage; for example, the RAD51 gene is vital for repair byhomologous recombination. The RAD51 gene product has
also been found to interact with products of the breast-cancer
susceptibility genes, BRCA1 and BRCA2, suggesting anunsuspected role for these genes in damage recovery. In
accordance with this finding, mice defective for the BRCA
genes have symptoms very similar to Rad51-defective mice,and the Brca2-defective mice are radiation-sensitive.
Additionally, some repair genes when knocked out in animals
give embryonic lethality, showing that these genes haveimportant roles in basic cellular processes influencing tissue
development.

Damage recognition[BRCA1/BRCA2]

ATM / ATR[PARP]
Mismatch repair
hMLH1
hMSH2

hRAD50 / hMRE11 / NBS1
DNA
replication

Homologous
recombination
repairhRAD51hRAD52
hRAD54XRCC2
XRCC3Nonhomologous
end joining

Repeatbased
end joining

Translesion
synthesis

XRCC4
hRAD52?
hREV3 (polymerase zeta)
XRCC5 (Ku86) ERCC1 / XPF? hRAD30 (polymerase eta)
XRCC6 (Ku70)
XRCC7 (DNA-PK )LIG4 (ligase IV)
C S

Base
excision
repairhNTH / hOGG1
HAP1
XPG
POL ß (polymerase beta)
XRCC1
LIG3 (ligase III)

POTENTIALLY MUTAGENIC PATHWAYS

Figure III. Important genes in DNA repair pathways in human cells following damage by ionizing radiation.
Some of the important genes identified in each pathway are shown (the h prefix indicates a human homologue of a yeastgene); names of proteins are shown in brackets if different from the gene names. The BRCA genes and the PARP gene(square brackets) have speculative roles in recombination repair and base-excision repair, respectively. Pathways that canlead to mutation are indicated.

18

ANNEX F: DNA REPAIR AND MUTAGENESIS

81. The conservation of repair genes from lower to higherorganisms and progress in understanding the human genome
suggest that further rapid progress will be made in the
discovery and analysis of genes influencing radiation
sensitivity. These genes will be important tools in understanding the extent of variation in radiation sensitivity in thehuman population and should help to identify individuals
especially at risk.

B. RELATIONSHIP BETWEEN REPAIR AND
OTHER CELL REGULATORY PROCESSES
1. Radiosensitivity and defective
recombination in the immune system:
non-homologous end joining of DNA doublestrand breaks
82. It has been noted that radiosensitive disorders are often
associated with some degree of immune deficiency. The recent
discovery that the scid (severe combined immune-deficient)mouse strain is radiosensitive has led to a reexamination ofthis relationship and to rapid progress in understanding atleast one mechanism of DNA-break rejoining. In the immune
system, functional immunoglobulin and T-cell receptor genes
are assembled from separate genetic regions during lymphoid
differentiation [A5]. The separate regions (termed "V" forvariable; "D" for diversity, and "J" for joining) involved inthis type of site-specific recombination are flanked byrecognition (signal) sequences at which double-strand DNA
breakage occurs prior to the rejoining of the regions. Micehomozygous for the scid mutation lack functional T-cells andB-cells because of a defect in V(D)J recombination [H8, L9],and about 15% of the mice develop lymphomas [C50]. It was
shown that fibroblasts derived from homozygous scid mice arethree to four times as sensitive as normal mice to x rays or
gamma rays and have a reduced ability to repair DNA doublestrand breaks [B8, H10]. Mice heterozygous for the scid
mutation also show some increased sensitivityto gamma rays,
compared to normal mice, and cultured bone marrow cells
from scid heterozygotes are marginally more radiosensitive
than normal cells [K45]. Detailed studies of repair capacity inscid cells show that the rate of repair of double-strand breaks
is reduced by a factor of about 5, but if sufficient time isallowed (24 h) the final levels of repair are similar to those of
normal cells [N20]. Subsequently, similar tests on several
radiosensitive mammalian lines derived from cultured cells
showed that those with large defects in double-strand breakrepair are also defective in V(D)J recombination, while
radiosensitive lines, including ataxia-telangiectasia cells, withnear-normal double-strand break repair are not V(D)Jrecombination-deficient (Table 2) [H12, K25, P6, T4, T11].
83. At least four different gene products are common to
V(D)J recombination and repair of radiation-induced DNA
breaks; three of these products are defined by theirrespective rodent sensitive lines: xrs, scid/V3, and XR1/M10 (Table 2). Recently there has been a breakthroughin identifying the genes and gene products complementing

the defects in these lines, to define a repair pathway termed
non-homologous end joining (paragraph 31). The xrscomplementing gene (also known as XRCC5) codes for asubunit of the Ku antigen (p86), a DNA end-binding
protein discovered in normal human cells that reacted with
sera from patients with certain autoimmune diseases [T5].
The scid/V3 protein (XRCC7 gene product) has been foundto be the catalytic subunit of a large DNA-dependent
protein kinase, also known as p460, belonging to the PI-3
kinase family and related to the ataxia-telangiectasia geneproduct (see paragraph 56) [H17]. The Ku antigen consistsof two subunits, p70 and p86, which interact to form adimer; this dimer binds to broken ends of DNA, recruiting
the p460 subunit and conferring kinase activity on thecomplex [G7]. The DNA-dependent protein kinase has
been shown to phosphorylate a number of substrates invitro, including the p53 tumour suppressor and RNA
polymerase II [A4], but it is not clear whether these
constitute important targets in vivo. The most recently
cloned gene of this series, the human XRCC4 genecomplementing the XR-1 line, encodes a protein unrelated
to any other yet described [L30]. Functional studies of the
XRCC4 protein show that it is a nuclear phosphoproteinthat is a substrate in vitro for the DNA-dependent protein
kinase and that it associates with the recently discovered
DNA ligase IV [C51, G30]. This discovery suggests that
XRCC4 acts as a go-between in the assembly of a DNAbreak repair complex in which the final step is mediatedDNA ligase IV (paragraph 66). Experiments using cell
extracts show that the rejoining of breaks by mammalian
DNA ligases is stimulated by purified Ku86 protein,especially when the break ends cohere poorly [R24]. Thisobservation suggests that Ku86 may function to stimulate
ligation, perhaps through its ability to bridge the gap
between broken ends. However, the precise mode of actionof these proteins in promoting the repair of DNA doublestrand breaks (and presumably in initiating a coordinatedresponse through kinase action on relevant proteins) ispresently under intensive study.
84. The different subunits of the DNA-dependent protein
kinase are not induced following x irradiation [J22]. However, the gene encoding Ku86 in primates (but not in rodents)
can be expressed in a form that has been found to respond toradiation damage. In this form the gene gives rise to a related
protein, termed KARP-1, which includes a p53 binding site[M43], and the RNA transcript and protein were increased bya factor of up to 6 at 90 min following x-ray irradiation.
Interestingly, this induction did not occur in cells defective ineither p53 or ATM, suggesting that at least some of the nonhomologous end joining is activated through interactions with
p53 and ATM signalling processes [M44].
85. Sequencing the entire gene encoding the p460 kinasefrom scid cells showed that a single DNA base alteration
leads to a premature stop codon in the highly conserved
terminal region of the gene [A20], as other data had predicted[B37, D11]. This mutation gives a protein truncated by 83
amino acids in scid cells, leading to a partial abolition ofkinase activity. Mutations in this gene have subsequently been

ANNEX F: DNA REPAIR AND MUTAGENESIS

found in other radiation-sensitive lines of cultured cells; forexample, the SX9 line (Table 2) has a mutation giving rise toa single amino-acid substitution at a position before that of thescid mutation, and leads to a more severe phenotype than thescid mutation [F25]. The partial loss of activity in scid [A19,
B38, C52, P26] mayexplain another surprising phenomenon,
whereby sublethal irradiation of scid mice can transiently
rescue V(D)J recombination [D12]. While the mechanism of
this rescue is still unknown, it suggests that p460 in scid is notcompletely inactive.
86. The importance of the catalytic subunit of DNAdependent protein kinase in the development of T-cells andB-cells and in protecting animals against cancer has been
illustrated by the chance finding of transgenic mice in which
the Xrcc7 gene was knocked out [J20]. These mice, namedslip, showed a lack of mature lymphocytes, but most remarkably all animals died within 5-6 months from thymic lymphoblastic lymphomas. No tumours of B-cell or myeloid linkage
were found, suggesting that Xrcc7 acts as a tumour suppressor of the T-cell lineage. Treatment of scid mice withgamma rays (1 Gy) at 24-48 h after birth led to 86%
developing T-cell lymphoma with very short latency, with noother tumour type observed [G35]. Further, mice having boththe scid defect and a knockout of the Parp gene (paragraph77) also develop veryhigh levels of T-cell lymphoma, suggesting that the carcinogenic effects of a partial defect in doublestrand break rejoining is exacerbated by a reduction in repair
capacity for other types of DNA damage [M6].
87. The assessment of radiation sensitivity of different
tissues has shown that epithelial cells of the intestine andkidney in scid mice show a much greater radiosensitization
than bone marrow cells from the same animals, comparedto the relative sensitivity of these cells in normal animals[H28]. However, the additional sensitivity of scid epithelial
cells effectively made these cells similar in overall sensitivity to scid marrow cells (e.g. at a dose of 2 Gy), sincenormal epithelial cells are more radioresistant than normal
marrow cells. Irradiation of epithelial cells at low dose rate
(16 mGy min-1) altered the survival of scid cells much lessthan it did for normal cells [H28], as expected for this typeof radiosensitive cell line (see paragraphs 49!50).
88. In addition to the scid mouse as a model of defective
DNA double-strand break repair, knockout mice for thegene encoding Ku86 have been created [N21, Z9]. These
mice weigh 40%-60% less than normal mice, and further
weight loss occurred when newborn mice were gammairradiated at whole-body doses in excess of 0.25 Gy.
Survival of Ku86-deficient mice irradiated at 2-4 months
old with gamma rays was also compromised; doses of 34 Gy caused 50% mortality in two weeks. Ku86-deficient
mice given 2 Gy gamma rays survived for up to 12 weeks
but unlike normal mice showed severe hair loss within one
month post-irradiation. Examination of tissues at four days
post-irradiation showed severe injury to the GI tract atmuch lower doses than for the normal mice, along withatrophy of lymphoid organs [N21]. In these mice it was
found that T- and B-cell development was arrested at an

19

early stage, as in scid mice. The Ku70 subunit has beenknocked out in mouse embryonic stem cells, and these cells
are sensitive to gamma rays. As might be expected, Ku70deficient cells are defective in DNA end-binding activity
and in V(D)J recombination [G31]. Ku70-deficient mice
have similar growth reduction and radiation sensitivity to
Ku86-defective mice, but they also develop a highfrequency of spontaneous thymic and disseminated T-cell
lymphomas [L57].
89. Mice lacking DNA ligase IV do not survive beyond thelate embryonic stage of development; ligase IV-deficientembryonic fibroblasts are hypersensitive to gamma rays but
not to ultraviolet light and are defective in V(D)J
recombination [F20]. Very similar defects have been found inmice and murine fibroblasts lacking the Xrcc4 gene. Further,lethality in XRCC4- and ligase IV-deficient mouse embryos
is associated with severe disruption of the development of thenervous system due to extensive apoptotic cell death [G33]. It
is presumed that the enhanced sensitivity of neuronal cells
relates to an inability to repair DNA double-strand breaks,
although it is not known whether these breaks result fromnormal metabolism or a specific neuronal process (e.g. a
recombination process) required for neuronal function. It is of
considerable interest that a single person defective in DNA
ligase IV has been identified (paragraphs 43 and 66); thisperson showed extreme sensitivity to radiation and developed
acute lymphoblastic leukaemia at age 14, but was not severely
impaired otherwise. Analysis of the ligase IV gene andprotein in cells derived from this person showed that ligase
function was not completely defective, presumably explaining
their relatively normal developmental progress [R38]. This
example of repair deficiency is important in revealing that
mutations leading to partial activity of the gene product may
be permissive for growth and development, but may have
undesirable consequences including the possibility of cancer
formation.
90. The search for human mutations in the XRCC4, 5, 6,
and 7 genes among patients known to be compromised in
immune functions is beginning to yield some candidates[C28, H26].
91. There is a difference between the “programmed”
double-strand breaks generated in the V(D)J recombination
process and those caused by radiation damage. The breaksat V(D)J sites have 5 -phosphorylated blunt ends and canbe rejoined by a DNA ligase without further processing[S6], while radiation-induced breaks are often not directly
ligatable because of extensive damage to the sugars andbases at the break sites (see Section I.B). Since, in the cell,the non-homologous end joining pathway is involved inrepairing both types of break, it may be suggested that theexact structure (and possibly complexity) of the breaks does
not influence their recognition by the proteins that initiate
the rejoining process. However, it seems likely that thecontext in which the break occurs will influence its repair;in yeast cells, three proteins involved in modulating
chromatin structure (Sir2, Sir3, and Sir4) interact with theKu70 protein and have a role in DNA break rejoining
7

20

ANNEX F: DNA REPAIR AND MUTAGENESIS[T41]. A defect in any of the three SIR genes led toincreased sensitivity to gamma radiation, providing thehomologous recombination pathway was inactive. Whilethe changes in chromatin structure brought about by the Sirproteins are not well understood, it is thought that they
may make the broken DNA inaccessible to other DNAmodifying enzymes such as nucleases and thereby protect
the damaged DNA from loss. It has also been found that
the V(D)J recombination process is restricted to the G1
phase of the cell cycle [S6]. It is likely that this rejoining
process is also cell-cycle-controlled in the repair ofradiation-induced double-strand breaks [L51]. Thus, the
XR-1 line is highly sensitive and defective in radiationinduced double-strand break repair in G1 but has nearlynormal sensitivity and break repair in late S phase [G5].
One implication of this analysis is that there must be atleast two major pathways for the repair of DNA doublestrand breaks: the non-homologous end-joining pathway
active in G1 and at least one other pathway operating inother stages of the cell cycle.

these two pathways gives a reduction in chromosomal
aberration frequency [F24]. This observation has been considered as evidence for the involvement of non-homologous
end joining mechanisms in chromosomal aberration
formation.

92. As noted in paragraph 70, there is evidence suggestingthat the homologous recombination repair pathway is active
in late S/G2 stages of the cell cycle; additionally, chick cells
defective in the RAD54 gene were found to be more sensitive
than normal cells in this part of the cycle. Chick cell lines
were generated with defects in both the homologous recombination repair pathway and the non-homologous end-joining
pathway (RAD54/Ku70-defective), and these showed
increased radiation sensitivity and higher levels ofchromosomal aberrations than either single defect [T42].

95. The rejoining of DNA double-strand breaks, produced
enzymatically, has also been examined using small DNA
molecules transfected into mammalian cells or exposed to cell
extracts. These studies have shown that almost any sort ofbreak end (with flush ends, or with complementary sequenceat the ends, or with mismatched ends) can be rejoined bycellular enzymes [N7, P29, R1]. However, even withcomplementary ends, a fraction of the breaks are rejoined witha loss of sequence around the break sites. This mis-rejoining
process was found to occur by a non-conservative
recombination mechanism [T10] that appears to differ fromthe non-homologous end-joining pathway. The mechanism
entails deletion of DNA bases between short (2-6 bp) direct
repeat sequences, such that one of the repeats is also lost
(Figure IV; see also paragraph 182). Occasionally the misrejoining can be more complex, with an insertion of DNA at
the deletion site. Using substrates that attempt to model moreclosely DNA double-strand breaks produced by radiation
damage, where the sequences at the break-ends are notcomplementary, breaks were shown to be rejoined by either
this repeat-driven mechanism or by a process of blunting theends before rejoining [M33, R18]. The repeat-driven processof mis-rejoining has been shown to be independent of the Ku
proteins [M33]. Recent data with yeast cells reveal that thismis-rejoining mechanism prevails where both the homologous
recombination and non-homologous end-joining pathways areknocked out [B35, B36]. This error-prone mechanism
constitutes, therefore, a third pathway for the rejoining of
double-strand breaks common to many organisms.
Interestingly, in extracts from two ataxia-telangiectasia celllines, the frequency of mis-rejoining by this mechanism wasabout 20 times higher than in extracts from normal cell lines[G3, N7]. The genetic basis of this short repeat-driven process
is unknown, although in principle it may require similar
enzymes to those that recombine adjacent homologous
sequences within a chromosome. Adjacent sequences withhomology of several hundred base pairs recombine in aprocess termed single-strand annealing in both mammaliancells and yeast; in budding yeast some of these recombination

93. Unlike mammalian cells, in lower eukaryotic organisms
such as the budding yeast, repair of DNA double-strand
breaks is mainly effected through homologous recombination
processes. However, proteins similar to the mammalian
Ku70/86 have recently been found in yeast, and these form adimeric complex binding directly to DNA ends. DNA endjoining in yeast was shown to be impaired when the genes
encoding the yeast Ku proteins are mutated, and this process
is distinct from the rejoining mediated by homologous
recombination. However, it is possible that some activities areshared by both homologous and illegitimate recombination
pathways, since it was found that the RAD50 gene product(paragraph 70) interacts with the yeast Ku proteins [M32].
Mutation of the gene encoding the smaller Ku subunit(HDF1) has also been found to lead to shortened telomeres,similarly to TEL1 mutations (paragraph 64), suggesting thatin addition to their end-joining role these proteins may help
protect yeast chromosome ends [P21]. Intriguingly, both the
Ku and Sir proteins are located at telomeres in undamaged
yeast cells, but following the induction of DNA double-strand
breaks they relocate to break sites in the genome [M54]. Lossof HDF1 gene function alone does not, however, lead toradiation sensitivity or a measurable defect in DNA doublestrand break repair in yeast, while the combined loss of HDF1
and homologous-recombination gene functions leads toextreme sensitivity [B35, S50]. Additionally, while the loss ofhomologous recombination repair in yeast leads to an elevatedfrequency of chromosomal aberrations, the combined loss of

94. While ataxia-telangiectasia cells are not defective in
V(D)J recombination, a recurrent feature of the disorder isthe appearance of T-cell clones with characteristic chromosomal rearrangements at sites of immunoglobulin andT-cell receptor genes [A7, B2, R4]. Elevated levels ofrecombination of T-cell receptor genes have been describedin lymphocytes from ataxia-telangiectasia patients relative
to those from normal individuals [L10]. ATM-deficient
mice develop thymic lymphomas and die by 5 months(paragraph 62), but if the gene that normally causes
programmed double-strand breaks leading to V(D)J
recombination is inactivated, no lymphomas develop [L66].
This striking observation suggests that ATM normallysuppresses aberrant recombination events.

ANNEX F: DNA REPAIR AND MUTAGENESIS

events are RAD52-dependent, but also require the products of
the RAD1 and RAD10 genes [O5]. RAD1 and RAD10
combine to give a single-stranded endonuclease required tosnip off the overhanging DNA strands generated by therecombination of adjacent repeats. In mammalian cells, thehomologues of RAD1 and RAD10 are the nucleotide-excision
repair genes ERCC1 and XPF, respectively [W52], and thereis evidence that the ERCC1 gene is involved inrecombination-dependent deletion formation in mammaliancells [S85].
Double-strand break
A

T

C

G

C

T

A

G

C

G

A

G

A

A

C

G

T

C

A

G

C

A

G

T

C

G

T

C

A

G

T

Exonuclease excision
A
T

A
A

G

C

Annealing of repeats
A
A
T

A

C

G

G

C

T

C
G
G
A

Ends snipped,
polymerase filling and joining.......

A

T

C

G

T

C

T

A

G

C

A

G

.......

Figure IV. Direct-repeat end joining model.
Direct repeats (shown in boxes) are identified on either sideof the initial double-strand break. Exonuclease removes
opposite strand beyond repeats. Repeats are annealed,leaving extended tails (snip ends), which are removed.
Strands are infilled by polymerase and ligated to give thedeleted form with only one copy of the repeat.

96. Ataxia-telangiectasia cell lines were found to haveelevated rates (by a factor of 30-200) of intrachromosomal
recombination in an introduced DNA construct [M8]. Thesefindings suggest that ataxia-telangiectasia cells have a general
disturbance in recombination processes, and that the cell
extract studies highlight those events leading to sequence loss
and rearrangement (see Section IV.B). It seems likely that anelevated level of non-conservative recombination would also
lead to an increase in mutation frequency, provided that the
mutation system (i.e. target genomic region) allows detectionof relatively large deletions and rearrangements. Evidence ofan increased spontaneous frequency of mutations in ataxiatelangiectasia relative to normal persons has been found fromdirect analysis of their blood cells. Measurements made in
ataxia-telangiectasia erythrocytes (loss ofheterozygosityin the
GPA system, paragraph 213) show loss mutations were
elevated, on average, by a factor of 10, while ataxiatelangiectasia lymphocytes (HPRT gene mutation) show anaverage elevation of about 4 [C15]. In each system, however,there was a wide range of frequencies, from nearly normal tomore than 100-fold higher than normal. These results are also
supported by studies of transfection of small target genes

21

carrying breaks at specific sites (by endonuclease cutting) on
shuttle vectors. The proportion of correctly rejoined target
genes was lower in ataxia-telangiectasia than normal cell lines[C13, R5, T6]. It is unclear whether this recombination defectis directly related to radiosensitivity in ataxia-telangiectasia,
but disturbance of recombination would tend to give genetic
instability and ultimately may lead to cancer-proneness.
2. Radiosensitivity and the cell cycle
97. Mammalian cells vary in their radiosensitivity throughthe cell cycle, generallyshowing greatest resistance during theperiod of DNA synthesis (S phase) and least resistance during
G2/mitosis and late G1 (where a long G1 exists) [S37]. To
account for this variation in response, different hypotheses
were advanced, such as fluctuation in radioprotective
substances in the cell and/or duplication of the genetic
material in S phase [S38, T27]. However, a number of studies
indicate that the fluctuation in response correlates to theability of cells to repair radiation damage when irradiated incertain cell-cycle phases [I3, W25]. Additionally, it has more
recently been found that certain DNA repair-defective celllines have altered cell-cycle responses (see paragraph 91).
These results suggest that the opportunityfor repair of damage
in relation to DNA synthesis or segregation is an important
determinant of response (see paragraph 9). Accordingly, theability of the cell to optimize repair and to avoid theinteraction of damage with other cellular processes bymodulating the cell cycle after irradiation may be crucial torecovery and genome stability.
98. It is now well established that some radiosensitive (rad)cell lines in yeasts are defective primarily in regulation of thecell cycle. The paradigm for this type of defect is the rad9 lineof budding yeast. Although identified many years ago by
sensitivity to x rays and UV light, it was discovered more
recently to be defective for a regulatory function (termed acheckpoint) in the G2 phase of the cell cycle. The loss of thischeckpoint allows rad9 cells carrying DNA damage toproceed into mitosis and die. The normal RAD9 gene productappears to respond only to damage, since rad9 mutant cells
have normal division kinetics when unirradiated, althoughtheydo show spontaneous chromosomal instability[W6, W7].
Irradiated rad9 cells have been shown to be defective also in
extended-G1 arrest, indicating that the normal RAD9 geneproduct operates at more than one transition point in the cell
cycle [S9].
99. Numerous other checkpoint genes have now been
identified, especially in lower eukaryotes such as yeasts, andhomologues of many of these regulatory genes are present in
human cells [C42]. It is clear, therefore, that all cells regulate
their cycling in relation to a variety of signals to make surethat a proper progression of phases occurs (e.g. that mitosis
does not occur until the S phase is finished) but also to ensurethat the genome is intact before starting a new phase. Certain
gene products, for example, the tumour suppressor p53,
appear to be central to this monitoring process in mammaliancells [A21, C43], and the detailed workings of this regulatory
network are currently the subject of intense research.

22

ANNEX F: DNA REPAIR AND MUTAGENESIS

100. It has been known for many years that x irradiation of
mammalian cells in extended-G1 phase (slowly proliferating
primary cultures or cells grown until they run out of nutrients["plateau phase"]) induces a dose-dependent G1 arrest [L7,
N1]. This arrest is evident at doses as low as 100 mGy, but the
duration of the arrest is reduced if the G1 cells are allowed
time to recover before restarting the cycle. Kastan et al. [K2]observed increases in the levels of the tumour suppressor
protein p53 correlated to a transient G1 arrest after relatively
low doses (500 mGy) of gamma radiation in normal humanhaematopoietic cells. Cells that were mutant for p53 showed
no G1 arrest but retained the G2 arrest found typically after
irradiation (but see also paragraph 109). The apparent
increase in p53 derives from a prolonged half-life of theprotein (the protein half-life is normally only about 30
minutes). The link between p53 and G1 arrest has been
generalized for other cell types and for a variety of DNAdamaging agents [F4]. A likely role for p53, given its
function as a transcriptional activator, is to activate the genes
involved in negative growth control. Radiation-induced G1arrest was found to correlate to inhibition of at least two
members of the compound family of proteins that controls
cell-cycle progression (the cyclin/cdk proteins); this inhibition
was dependent on p53 and was mediated by the p53-inducible
kinase inhibitor p21 [D4].
101. The signal for p53-dependent G1 arrest is likely to be
DNA breakage; agents causing DNA double-strand breakage(ionizing radiation, bleomycin, DNA topoisomerase
inhibitors) are very effective inducers, while UV light appears
to cause p53 induction through the processing of base damage
into breaks. The introduction of restriction endonucleases into
cells has shown that DNA double-strand breakage alone issufficient for this response [L14, N5]. Microinjection of
broken DNA molecules into cell nuclei has suggested thatvery few double-strand breaks may be required for arrest, andonly one or two may be sufficient [H29].
102. Ataxia-telangiectasia cells lack DNA-synthesis arrest(Section II.A.1) and G1 arrest, and the DNA-damaging agentsthat strongly induce p53 are also the agents to which ataxiatelangiectasia cells are sensitive [L14, N5]. It has further beenshown that p53 induction is reduced and/or delayed in
irradiated ataxia-telangiectasia cells and suggested that theataxia-telangiectasia gene product (ATM) is part of asignalling mechanism that induces p53 [K3, K6, L14]. A
similar alteration of p53 response has been found after irradiation of cells derived from patients with Nijmegen breakage
syndrome [J21]. ATM has been shown to physically associatewith p53; it is responsible for a rapid phosphorylation of a
specific residue at the N-terminal end of p53; this phosphorylation occurs within minutes of treatment of cells withionizing radiation or radiomimetic drugs but not after UVlight treatment [B59, C58, K43, S73]. These processes
contribute to its increased half-life following irradiation andalso lead to the association of p53 with other proteins involved
in damage-signalling pathways. In mouse fibroblasts deficientin both ATM and p53 proteins (or ATM and p21), the loss of
the G1/S cell-cycle checkpoint following irradiation is noworse than for either single defect. This again suggests that

ATM and p53 operate in a common pathway of cell-cycle
control in response to DNA damage. However, it seems that
ATM may regulate only some aspects of p53-dependent
responses; for example, the ATM defect leads to prematuresenescence in mouse fibroblasts, and in this case the combined
deficiency of ATM and p53 [W47] or ATM and p21 [W49]alleviates the senescence.
103. The ATM protein has also been shown to interact
directly with the proto-oncogene c-Abl [B48, S65], a protein
kinase that itself interacts with other important cell-cycle
regulators such as the retinoblastoma protein and with RNA
polymerase II. When cells are irradiated, c-Abl is activated innormal cells, but this activation is absent in ataxiatelangiectasia cells. Additionally, cyclin-dependent kinases areresistant to inhibition by radiation in ataxia-telangiectasia
cells, and this appears to be due to insufficient induction of
p21 [B30]. A kinase named CHK1 that links DNA repair withcell-cycle checkpoints in yeast, has recently been found to beconserved in humans and mice and is dependent on ATM for
its activity in meiosis. Like ATM (paragraph 62), CHK1
protein localizes along synapsed meiotic chromosomes,
probably at the time that they are repairing DNA breaks. It is
speculated that CHK1 acts to coordinate signals from both
ATM and ATR (paragraph 65) to ensure the correct
progression of meiosis [F17]. The activity of a second kinase,
CHK2, which is homologous to the yeast RAD53 protein(paragraph 70), has also been shown to be ATM-dependent;
this kinase is involved in the negative regulation of the cell
cycle [M23]. It seems likely, therefore, that the ATM protein
is involved in multiple signal-transduction pathways, byvirtue
of its interaction with a number of important regulatory
proteins.
104. Alterations in the response of cell-cycle regulators in
ataxia-telangiectasia cells may be involved in some aspectsof the disorder but are unlikely to be responsible for theradiosensitivityof ataxia-telangiectasia. For example, p53deficient cells have a tendency to be radioresistant rather
than radiosensitive [L6], and SV40-transformed ataxiatelangiectasia cell lines in which p53 function is ablated
still have the characteristic radiation sensitivity. It is more
likely that any compromise of p53 response will affectcancer-proneness; both human patients (Li-Fraumeni
syndrome) and Tp53-knockout mice show elevated levelsof cancer [D3, M4]. Loss of one (Tp53+/-) or both (Tp53-/-)gene copies in mice also has significant effects on
sensitivity to radiation-induced cancers, seen as a much
reduced time for tumour onset. When Tp53+/- mice were
irradiated with gamma rays to a dose of 4 Gy at 7-12
weeks, the tumour onset time was reduced from 70 to 40
weeks, while Tp53-/- mice showed a decreased latency only
if irradiated when newborn (because at later times theirfrequency of spontaneous tumour development was alreadyextremely high, masking the effect of irradiation). It is ofinterest that p53-deficient mice show a high incidence ofthymic lymphomas, similar to ATM- and BRCA2-deficient
mice (paragraphs 62 and 73), although it should be
remembered that this result may be influenced by the factthat all of these mice were produced in the same genetic

ANNEX F: DNA REPAIR AND MUTAGENESIS

background. In the Tp53+/- mice, the normal Tp53 allele
was commonly lost in tumour cells; it was notable that inradiation-induced tumours there was both a high rate ofloss of the normal allele and a duplication of the mutant
allele [K11].
105. Inactivation of mitotic checkpoint genes has beenfound in tumour cells derived from both BRCA1-deficientand BRCA2-deficient mice (paragraph 73), suggesting thatthese defects cooperate in cancer progression. Conditionaldeletion of the Brca1 gene led to a low frequency and longlatency for mammary tumours, but Tp53+/- mice carrying
this Brca1 mutation showed a significant acceleration in
mammary tumour formation [X7]. It may be significant
that the phosphorylation of BRCA1 in response to DNA
damage is mediated by ATM [C67], in view of thepotential involvement of ATM in breast cancer (paragraph
137 et seq.). In BRCA2-deficient cells, mutations in Tp53
or genes controlling the mitotic checkpoint were implicatedin promoting cellular transformation and the developmentof lymphomas [L64]. Centrosomes, which control the
movement of chromosomes during mitosis, are abnormal
in both BRCA1- and BRCA-2-deficient cells, leading tounequal chromosome segregation [T46, X8].
106. Mice that lack DNA-dependent kinase (scid phenotype,paragraph 82) accumulate high levels of p53 because of thepresence of “natural” DNA double-strand breaks from
unrepaired V(D)J recombination intermediates but show atypical G1 arrest following irradiation. These mice develop
thymic lymphoma with low incidence (15%) and long latency.
The importance of p53 expression in protecting the animals
from cancer was shown in mice that lack both DNAdependent kinase and p53; these mice show prolonged
survival of lymphocyte precursors and onset oflymphoma/leukaemia by 7-12 weeks of age (mice defective in
p53 alone develop lymphoma to 50% incidence at 16-20
weeks). Cell lines derived from these double knockout mice
were also about 10 times more resistant to gamma radiation
than lines from scid mice. It was suggested that p53 may
normally limit the survival of cells with broken DNA
molecules and therefore that p53 loss promotes genetic
instability [G23, N14].
107. Many of these p53-related responses may be associatedwith other functions of p53, in particular its role in radiationinduced apoptosis (programmed cell death, Section II.B.3). It
is clear that DNA- break-inducing agents, including ionizing
radiation, kill some cell types through a p53-dependent
apoptotic pathway, although this pathway is not involved in
the induction of apoptosis by other stimuli [C6, L13].
Alterations in the control of apoptosis may be linked tocarcinogenesis, where a loss of p53 function may lead to thesurvival of precancerous cells, as has been shown in the
induction of skin cancer by UV light [Z4].
108. It has also been known for many years that irradiatedmammalian cells may arrest in G2. The G2 checkpoint is also
controlled by specific cyclin/cdk proteins, and gamma
radiation rapidly inhibits the kinase component (p34cdc2) at

23

doses resulting in G2 arrest [L12]. Conversely, p34cdc2
activation accompanies release of a radiation-induced G2
block by drugs such as caffeine [H14]. In cultured cells,
caffeine-induced release of the arrest decreases survival [B49]
and increases the proportion of cells dying from apoptosis[B40]. However, it has also been found that caffeine treatment
leads to an increased frequency of chromosomal aberrations[L40, T26], although the link between these two effects has
been questioned [H18]. These observations suggest that thearrest promotes survival and may reduce the probability ofgenetic alterations. This view is supported by experimentswith arrest-defective rad9 yeast cells, where the imposition ofan artificial G2 delay promotes survival [W6]. However, insome radiosensitive mammalian cell lines, including ataxiatelangiectasia, enhanced delay in G2 is commonly linked toenhanced sensitivity [B3, B6]. It seems likely that because of
repair defects these cells never progress further in the cycleand die (that is, the cell-cycle arrest is irreversible, because thedamage signal is not reduced). The signal for this response
could be unrepaired DNA breaks; recent experiments inrecombination-deficient yeast carrying a single unrepaired
double-strand break in an inessential DNA molecule showthat this can be lethal to a majority of cells [B7].
109. Initially there was little evidence of a role for the p53
protein in G2 arrest (paragraph 100), but more recent datawith p53-deficient mice and cells have shown that p53 isrequired for correct control of entry into mitosis following
DNA damage [B60, S74, S75]. Similarly, cells establishedfrom patients with germ-line TP53 mutations (Li-Fraumeni
syndrome) have a consistent defect in G2 response [G36].
3. Apoptosis: an alternative to repair?
110. Early studies of irradiated mammalian cells recognizedthat there are different forms of cell death [O1]. Attempts tocorrelate cell death with the formation of chromosomal
aberrations concluded that a major cause of death infibroblastic cells is genetic damage resulting in the loss ofchromosome fragments [B22, C26, D8]. The most conclusive
experiments were those of Revell and co-workers, showing aclose correlation between loss of chromosome fragments,
observed as micronuclei, and cell death in individual diploid
Syrian hamster BHK21 cells [J13]. It is envisaged in thesecells that genetic imbalance, perhaps due to the loss of specificessential genes, results in death once the pre-irradiation levelsof their gene products have decayed. However, cells from anumber of different developmental lineages are known toundergo rapid cell death, often termed interphase death, after
irradiation [H16, P18, Y4].
111. It is now recognized that the balance between cellproliferation and cell death is crucial to the correct
development of organisms, and that cell deaths in manytissue lineages are programmed bya genetically-controlled
process known as apoptosis. This process was firstdescribed as a characteristic breakdown of cellular
structures, including DNA degradation [K14, W33].
Apoptosis is initiated through specific cell surface
receptors in response to external developmental signals but

24

ANNEX F: DNA REPAIR AND MUTAGENESIS

may also be induced by DNA damage. Radiation-induced
interphase death is considered to be an example ofapoptosis [A3, U14, Y3], although the occurrence of
apoptosis has more recently been reported in cell lines
dying at various times after irradiation [R8, Y5].
112. Initiation of rapid apoptosis in normal cells by radiation
appears to be dependent on the induction of the p53 protein[C6, L13], although delayed apoptosis may occur in cell lines
deficient in wild-type p53 protein [B27, H37]. While p53dependent apoptosis does not involve the same receptors as
developmentally-regulated apoptosis, thereafter the molecularpathways converge to a common cell death programme. The
mechanism of signalling by p53 is not well understood,
although p53 can bind DNA at specific sites (“response
elements”), and this binding is strongly stimulated by DNA
ends, short regions of single-stranded DNA, and shortmismatched DNA segments [J9, L28]. The importance of thisDNA binding is demonstrated by the fact that the vast
majority of TP53 mutations identified in tumour cells occur inthe part of the gene specifying DNA-binding activity [H25].
Relatively stable binding to DNA (half-life >2 h) can allow avariety of subsidiary processes to occur, such as the activationof specific genes. Because p53 has the properties of atranscriptional activator, it may initiate apoptosis byswitching on a specific set of genes. Alternatively there isevidence that in some situations p53 may act in the opposite
way, repressing genes concerned with cell survival [C23,C32].
113. A second pathway of radiation-induced apoptosis is
proposed to work through a membrane-associated signallingsystem responding to a variety of extracellular stresses.
Sphingomyelin and possibly other sphingolipids at themembrane respond to stress by rapidly releasing ceramide,
which accumulates and activates protein kinases to initiate
cell-cycle arrest and apoptosis. It has been suggested that
ceramide acts as a "biostat", measuring and initiating responseto cell stresses in the same way as a thermostat measures andregulates temperature [H34]. Radiation can activate components of the ceramide pathway in isolated membranes,suggesting that DNA damage is not required [H35]; however,this suggestion has been challenged [R39], and recently it has
been found that another function of the ATM gene is tomodulate ceramide synthesis following radiation-induced
DNA damage [L67]. Lymphoblasts from patients with the
Niemann-Pick disorder have an inherited deficiency in acidsphingomyelinase, an enzyme hydrolysing sphingomyelin togive ceramide, and fail to show ceramide accumulation andapoptosis following irradiation. Comparison of mice with aknockout in the gene for acid sphingomyelinase with Tp53knockout mice shows that the initial stages of thesphingomyelin-dependent apoptotic pathwayare distinct fromthose of p53-mediated apoptosis. These two pathways may beprevalent in different tissues; sphingomyelin-dependent
apoptosis prevails in the endothelium of the lung and heart
and the mesothelium of the pleura and pericardium, while
thymic apoptosis is especially dependent on p53. The acidsphingomyelinase-deficient mice develop normally, showing
also that this apoptotic pathway is distinct from develop-

mentally-regulated apoptosis [S55]. In tumour cell lines where
TP53 is mutated, it has also been shown that resistance toradiation correlates to loss of ceramide accumulation [M1].
114. Whether or not the cell goes into apoptosis in response
to a given radiation dose is thought to depend on theavailability of other proteins that promote or inhibit further
steps in the programme. The details of these steps are still
being worked out, but it is clear that a diversity of factors
influences apoptosis. For example, cells may become
susceptible to radiation-induced apoptosis through functionalloss of the retinoblastoma (Rb) protein, a cell-cycle regulator,
by either mutation of the RB gene or the expression of viral
oncoproteins that inactivate Rb [S42]. Conversely, the geneproduct of the BCL-2 oncogene, first identified at a site ofchromosome translocation in B-cell lymphomas, can blockradiation-induced apoptosis [R10]. It has been found that
BCL-2 and related proteins localize to cellular membranes,
especially of mitochondria, where permeability transitions areimportant in regulating the apoptotic process [G25]. The
competitive formation ofprotein complexes bymembers of the
BCL-2 family is suggested to control susceptibility toapoptosis; some of these proteins are antagonists, like BCL-2,
while others are agonists, and the relative proportions of these
proteins determine whether a cell will respond to an apoptotic
signal [K5, K19, W34].
115. The extent of apoptosis for a given radiation dose does
not appear to be affected by changes in the dose rate or bydose fractionation [H38, L26, L32], although there has beena recent report of a reduction in apoptosis at very low dose
rates from gamma rays (<1.5 mGy min-1) [B66]. There is aneffect of cell-cycle stage on the extent of apoptosis; in contrast
to the "classical" pattern of resistance (Section II.B.2),
mammalian cells appear to be resistant in G1 and sensitive in
S phase [L26, L33]. In cells that are relatively resistant toapoptosis, the oxygen enhancement ratio was found to be
similar for apoptosis and for clonogenic cell survival [H36].
The relative biological effectiveness (RBE) of fast neutrons for
apoptosis in thymocytes was found to be 1 [W16], in
accordance with earlier measurements of interphase death inthymocytes and lymphocytes [G15, H19]. However, forintestinal crypt cells of mice, where there is a highly sensitive
subpopulation of cells in the stem-cell zone, the RBE for
apoptosis was 4 for 14.7 MeV neutrons and 2.7 for 600 MeV
neutrons [H38, H39]. It is not known why the RBE varies fordifferent tissues; both thymocytes and intestinal cells show
p53-dependent apoptosis [L13, M39], but it is possible that
their relative abilities to repair radiation damage differ (seeparagraph 87). The proportion of cells dying from apoptosis,
relative to other forms of death, was shown to be constant at
different doses (range 2-6 Gy) in cells where the maincause of death was apoptosis [L26]. This result does notsuggest, at least for doses in excess of 2 Gy, that cells
switch on the apoptotic pathway because their repair
systems are unable to cope with the level of damage. In
human lymphocytes, significant induction of apoptosis by
gamma rays could be measured at doses as low as 50 mGy,and some evidence was found that individuals vary in theirapoptotic response [B41].

ANNEX F: DNA REPAIR AND MUTAGENESIS

116. It has been suggested that alterations in the p53dependent apoptotic pathway play an important part in thesensitivity of ataxia-telangiectasia [M38] and Fanconi s
anaemia [R20, R34], but this view has not been supported
by other studies [B51, D15, E15, K35, T43]. It has beenfound that p53-mediated apoptosis is normal in irradiatedAtm knockout mice [B50], but that ATM is involved in theregulation of the ceramide-dependent pathway [L67].
Further study of apoptotic responses, especially p53independent pathways, are required to clarify these
responses, preferably in primary cells from these disorders.
7

4. Summary
117. A link between radiation sensitivity and immune
system defects has been revealed from studies of DNA
repair genes; the same gene products are used to assemble
functional immune genes and to repair radiation-induced
DNA breaks by non-homologous end joining. Cells andanimals defective in genes in this pathway are extremely
sensitive to ionizing radiation and lack low-dose-rate
sparing. While non-homologous end joining seems to be
the main mechanism in mammalian cells for DNA doublestrand break rejoining, at least two other pathways exist,
and in lower organisms double-strand breaks are mostly
repaired by the homologous recombination pathway.
118. An important component of the non-homologous endjoining pathway, the XRCC7 gene encoding the catalytic
subunit of DNA-dependent kinase, is related in structure to
the ataxia-telangiectasia gene product and has been shown tobehave as a tumour suppressor. The mechanism of rejoining
by this pathway and the relationship of the components of thepathway to other regulatory processes (such as cell-cycle
control) are at present the subject of intense research activity.
119. It is clear that the fidelity of rejoining by the different
repair pathways is an important determinant of the mutagenic
consequences of DNA damage (see also Figure III).
Homologous recombination repair is likely to be the only
error-free pathway. However, since even this pathway
depends on copying information from another DNA strand,
the fidelity must relate to the quality of both the information
copied and the enzyme (polymerase) used for the copying.
Pathways of double-strand break repair based on illegitimate
recombination (non-homologous end joining, repeat-driven
rejoining) will commonly be error-prone, although inprinciple the non-homologous end joining pathway can beerror-free where the nature of the damage allows this (e.g.
repair of a double-strand break formed by the overlap of two
single-strand breaks sufficiently separated to allow for
templated repair). Repeat-driven rejoining leads to deletion
formation (mis-rejoining), and may act as a back-up fordamage that is not (or cannot be) repaired by the otherpathways. It is still not clear which factors determine the25

likelihood that a specific damage site will be repaired by aparticular pathway [T44]. The complexity of damage may
influence this question (Section I.B), as well as the possibility
that radiation damage induces the activity of specific repair
enzymes or pathways (Section III.B). In future work it will be
important to establish the relative efficiencies ofthese different
pathways at different doses and dose rates and in different cell
types, so that mutagenic consequences can be predicted. The
potential consequences of error-prone repair pathways at low
radiation doses are explored further in Annex G, “Biologicaleffects at low radiation doses”.
120. When growing cells are irradiated, they arrest theircell-cycle processes, apparently to allow time for repair tobe completed satisfactorily. This arrest is part of acheckpoint function that monitors the physical state of
DNA at different stages of the cycle. Cells that have lost acheckpoint may be as radiation sensitive as cells that havelost DNA repair capability. Many genes are involved incontrolling the cell cycle and determining checkpoints. In
mammalian cells, the p53 protein is important in response
to DNA breakage and controls both arrest in the G1 phaseand one pathway of programmed cell death (apoptosis).
Animals and humans (Li-Fraumeni syndrome) deficient in
p53 show elevated levels of cancer; irradiation of p53deficient mice has a marked effect on the latency period fortumour formation and gives a high incidence of thymic
lymphomas. The ataxia-telangiectasia disorder has defects
in cell-cycle checkpoint functions, and there is evidence
suggesting that ATM and p53 operate in a common
pathway of cell-cycle control in response to DNA damage.
121. Apoptotis (programmed cell death) is also induced byionizing radiation, both through the p53-dependent pathway
and a membrane-associated signalling pathway. The relative
importance of these pathways varies in different tissues; also,
the importance of apoptosis as a mechanism of cell death inresponse to radiation varies with the cell type anddevelopmental stage. The relationship between apoptotic deathand radiation injury differs from that for genetic death (loss ofessential genes through damage to the genome). Changes indose rate or dose fractionation do not appear to affectapoptotic responses, and the response of cell-cycle stage is thereverse of the pattern found for genetic death. While it isattractive to consider that in the face of excessive damage,radiation-induced apoptosis is an alternative to DNA repair,the evidence for this possibility is not convincing. Loss ofability to respond to apoptotic stimuli will allow theaccumulation of cells that may carry genetic damage and cantherefore be a cancer-promoting event [W8]. Thisphenomenon is seen, for example, in Tp53-knockout mice(paragraph 104) and in mice overexpressing the BCL-2
protein [M21]. In this respect, the correct functioning of
apoptotic pathways can be viewed as a complementarymechanism to the repair of DNA damage, removing damaged
cells from the population.

26

ANNEX F: DNA REPAIR AND MUTAGENESIS

III. HUMAN RADIATION RESPONSES
A. CONTRIBUTION OF MUTANT GENESTO HUMAN RADIOSENSITIVITY
122. The term radiosensitivity is used to indicate anabnormally increased response to ionizing radiation of both
the whole body (see Section II.A.1) and cells derived frombody tissues. Further, in cells the radiation response may bemeasured in different ways. For example, the ability of cells
to grow and form clones (cell survival) following irradiationis commonly used as a measure of sensitivity, but moresensitive assays based on chromosome damage may also be
used. In the comparative measurement of cell survival, theterms D0 (mean lethal dose calculated as the inverse of theslope for exponential survival curves represented semilogarithmically), Dbar (mean lethal dose calculated as the area
under the survival curve in linear representation [K34]), or
D10 (dose to give 10% survival) are commonly used.
123. The range of sensitivity of cells from supposed normal
individuals has been measured in survival experiments
following multiple doses of acute low-LET radiation. In two
major studies with human diploid fibro-blasts, the sensitivity
to dose was found to vary by a factor of about 2: D0 range =0.98-1.6 Gy (mean = 1.22±0.17 Gy) in 34 cell lines tested[C8] and D0 range = 0.89-1.75 Gy (mean = 1.23 ± 0.23 Gy)
in 24 cell lines [L8]. The degree of interexperimental variation
for a given cell line was generally small (#20%), although in
one of these studies cell lines derived from a mother anddaughter showed survival curves that could also varybya dose
factor of about 2 on repeated testing [L8]. However, it was
concluded that neither cell culture conditions (includingcloning efficiency and population doublings) nor age and sexof the donor were correlated to the observed differences inradiosensitivity [C8, L8]. This suggests that unknown genetic
factors that affect radiosensitivity vary in the cells of normal
individuals.
124. More recently it has also been possible to measure cell
survival with peripheral blood T-lymphocytes, irradiated as
resting (G0 phase) cells, to assess the range of radiosensitivity
in humans. G0 lymphocytes tend to have a more curvilinear
response to dose than cycling fibroblasts and, on average, ahigher survival at low doses than fibroblasts. Early-passage
fibroblast survival data generally show a good fit to a simple
exponential, while the G0 lymphocyte data are better fittedwith models including a dose-squared term, such as thequadratic (-lnS = αD + βD2). Using D0 as a measure, therange of sensitivity to dose of lymphocytes from different
individuals can also vary by a factor of about 2 [E7]; however,given the shape of the survival curves, D0 is difficult tomeasure accurately, and the range is generally much less than
2 using measures such as Dbar or D10 [E7, G17, N4].
125. No correlation was found between the measured
sensitivities of fibroblasts and lymphocytes from the same
individual [G17, K23]. A similar lack of correlation was

found between lymphocyte and fibroblast responses ofpretreatment cancer patients, whether the fibroblasts werecycling or in the plateau phase, suggesting that thesedifferences in response were not cell-cycle-dependent [G11].
The differences between fibroblasts and lymphocytes may
reflect several other possibilities: modes of death may differ,cell-type-specific factors may affect the expression of genesthat modify radiation response (especially after growth inculture), and/or there may be unknown variables in assay
conditions. In support of the last possibility, Nakamura et al.
[N4], using donors from one ethnic group, found a similar(relatively small) variation in the mean x-ray dose to kill 28
samples of T-lymphocytes from one individual (D10 =3.66±0.21 Gy), very similar to results with samples from 31
different individuals (D10 = 3.59±0.18 Gy). Elyan et al. [E7]reported similar data with donors from three ethnic groups,
although with a greater overall range of variability. In supportof differences in cell-type-specific factors, other cell types suchas keratinocytes also show survival that is significantly
different from that of fibroblasts taken from the same
individuals [S39].
126. The use of experimental conditions promoting recovery
from irradiation, such as low-dose-rate irradiation, will, it issuggested, expand the range of sensitivities to dose shown by
normal human cells. The exposure of non-growing fibroblasts
from 14 different normal individuals to tritiated water (doserate = 8.5-100 mGy h-1) expanded the range of sensitivity todose by a factor of more than 3 [L35]. Similarly, Elyan et al.
[E7] found that low-dose-rate irradiation (9.8 mGy min-1) of
human G0 lymphocytes from 19 individuals expanded therange of sensitivity to dose by a factor of about 4. When ahigher dose rate (28.5 mGy min-1) was used, however, there
was little difference between the range of sensitivities
measured for fibroblasts and lymphocytes and the range witha dose rate of 4.55 Gy min-1 [G11]. The latter result probably
reflects incomplete recovery during the low-dose-rateirradiation period [E7, G11].
127. Other assays of cellular sensitivity have beendeveloped based on the measurement of responses such as
the growth of cells after single doses of radiation. A"growback" assay following gamma irradiation oflymphoblastoid cells, developed by Gentner and Morrison[G4], detected a wide range of sensitivity in the normal(asymptomatic) population. Of 270 lymphoblastoid lines
tested, about 5% of normal lines showed hypersensitivity
after acute irradiation, as measured by their overlap withthe responses of ataxia-telangiectasia homozygotes, whileon the same criterion about 12% were hypersensitive whenassayed using chronic irradiation [G4, G13]. However, awider range of sensitivity was detected using chronic
irradiation, and rigorous statistical tests found that the
proportion of lines showing a significantly hypersensitive
response (p = 0.05) was 5%-6% under both irradiation
conditions [G14].

ANNEX F: DNA REPAIR AND MUTAGENESIS

128. There have been claims that a number of disorders
with an increased incidence of cancer are hypersensitive tox rays, including retinoblastoma, basal-cell naevus
syndrome, Gardner's syndrome, and Down's syndrome[A6, M2, W3]. The main method of determining sensitivity
differences has been cell survival assays with cultured
fibroblasts (as shown in Figure II), in which small numbersof cell cultures derived from normal individuals arecompared with those of one or more derived from thepatient(s). Unfortunately, such studies have not always
given consistent sensitivity differences for the same
disorder tested in different laboratories. In part this may be
due to the large range of sensitivity found among normal
individuals; if the normal lines used in a given laboratory
are in the higher part of the survival range, a disordershowing relatively low survival may be classified assensitive even though it falls within the overall normal
range. However, it has been concluded from a statisticalanalysis of many published survival curves of humanfibroblasts that the radiosensitivity of cells from somedisorders, calculated as Dbar, can be discriminated from thatof normal cells [D7]. The disorders include ataxiatelangiectasia homozygotes and heterozygotes, Cockayne's
syndrome, Gardner's syndrome, Fanconi's anaemia, and 5oxoprolinuria homozygotes and heterozygotes.
129. A number of studies had previously suggested that cells
from individuals heterozygous for the disorder ataxiatelangiectasia (carriers with one mutated gene, such as theparents of those showing the full symptoms) are moresensitive than normal to the lethal effects of irradiation [A6,
A14, C33, K17, P11]. Additionally, ataxia-telangiectasia
heterozygotes show an average level of dose-rate sparing thatis intermediate between that oflines from ataxia-telangiectasia
homozygotes (paragraph 49) and normal persons [P12, W18,
W21]. However, even under low-dose-rate conditions, where
a number of carriers have been tested, it is evident that their
sensitivity range overlaps that of the most sensitive normal
individuals [L35, W18]. Survival assays on cells derived fromhuman sources cannot therefore unequivocally distinguishcarriers of a mutated AT gene from normal individuals.
Similarly, it has been found that protracted radiation exposure
will not readily distinguish other disorders suggested to haveminor degrees of hypersensitivity [L35]. Inbred mouse strains
may show less variation in response from individual toindividual, and mice heterozygous for a defective Atm gene
were found to show a significant reduction in lifespanfollowing irradiation (4 Gy at 2-4 months of age), as well as
premature greying of coat colour, compared to normal
littermates [B61].
130. The finding of a range of sensitivities to radiation
among supposedly normal persons implies that in addition to
those with increased sensitivity, there are individuals withgreater radiation resistance than average. There is little
evidence, however, for specific disorders associated withradiation resistance. It has been noted that cell lines derived
from mice deficient in the p53 tumour suppressor are moreresistant to radiation than normal (paragraph 104), although
this response may be cell-type-dependent [B43]. Similar

27

studies on fibroblasts derived from patients with the Li-Fraumeni syndrome, defective in p53, highlighted a family
carrying a germ-line TP53 gene mutation in codon 245. Cells
from familymembers showed a radioresistance (D0 value) thatwas increased by a factor of 1.2 [B44], compared to an approximately 1.5-fold increase for the p53-deficient mice [L6].
131. There is evidence from cell survival assays thatradiation sensitivity at the cellular level may correlate withtissue response of cancer therapy patients. In a study of 811
patients treated with radiotherapy for breast cancer, five
showed severe skin responses. The survival of fibroblast
cell lines derived from these five patients was compared to
six lines derived from women with normal radiotherapy
responses (also from the same group of 811 patients). The
sensitivity of the severe-response patients (D0 =0.97±0.11 Gy) was significantly higher than that of thenormal group (D0 = 1.16±0.08 Gy; see paragraph 123)
[L39]. In a prospective study of 21 head-and-neck cancer
patients it was found that the sensitivity of their cultured
fibroblasts, but not their lymphocytes, correlated withwhole-body late effects [G12]. Similarly, a study offibroblasts derived from 10 radiotherapy patients showeda correlation between cellular sensitivity and late tissue
reactions [B42]. The survival of lymphocytes from breast
cancer patients suffering severe reactions to radiotherapy
has also been studied in conjunction with ataxia-telangiectasia heterozygotes and homozygotes [W21]. Using highdose-rate irradiation (1.55 Gy min-1), the survival of
lymphocytes from severe-response patients could not be
distinguished from that of normal-response patients (orother normal controls), but with low-dose-rate irradiation(9.8 mGy min-1) the cell survival of severe-response
patients and ataxia-telangiectasia heterozygotes was
similar and significantly different from that of normal
individuals. Using the growback assay (paragraph 127),
lymphoblastoid cell lines derived from newly presented,
non-selected cancer patients showed a similar range ofradiosensitivities to the normal lines, while patients alreadyknown to show some adverse reaction to radiotherapy
showed a higher proportion with extreme sensitivity[G14].
132. Assays based on chromosomal damage have commonly
shown a higher degree of sensitivity in the detection ofdifferences between individuals. For example, the genetic
disorders ataxia-telangiectasia, Bloom's syndrome, andFanconi's anaemia were described as “chromosomal breakage
syndromes” in early studies, showing significantly increased
frequencies of chromosomal breaks and exchanges after
irradiation [H24]. Chromosomal responses after low-dose-rateirradiation have also been used to examine the sensitivity of
ataxia-telangiectasia heterozygotes [W13] and breast cancer
patients who react severely to radiotherapy [J12].
Lymphocytes from 5 out of 16 patients (31%) sustaining
severe reactions to radiotherapy had dicentric yields outside
the control range [J12].
133. An assay based specifically on the chromosomal
sensitivity of lymphocytes to irradiation in G2 has beensuccessful in detecting significant differences between

28

ANNEX F: DNA REPAIR AND MUTAGENESIS

individuals carrying various cancer-prone disorders. The
work of Sanford et al. is based on the measurement ofchromatid breaks and gaps a short time after irradiation oflymphocyte cultures; this assay detected enhanced
sensitivity in fibroblasts from a number of disorders,
including ataxia-telangiectasia [T25], Fanconi's anaemia,
Bloom's syndrome, Gardner's syndrome, basal-cell naevus
syndrome, xeroderma pigmentosum, and familial polyposis[P2, S5]. In this G2 assay, cells from cancer patients andtumour cells showed an even higher frequency ofchromatid damage [H21, P3]. This assay has sometechnical difficulties; extensive tests in another laboratory
using identical conditions failed to reproduce many of theresults, which led to a modified assay that appears to be
more encouraging [S28].
134. The modified G2 chromosomal assay devised by Scottet al. [S27] has been used to assay the sensitivity oflymphocytes from 74 normal individuals, 28 ataxiatelangiectasia heterozygotes, and 50 breast cancer patients.
The ataxia-telangiectasia heterozygotes were clearly
distinguished in sensitivity from the majority of normal
individuals, but about 9% of the normal individuals
showed hypersensitivity. Human fibroblast cultures show
a similar result, with four ataxia-telangiectasia heterozygotes distinguished from seven normal individuals[M47]. The range of sensitivities of lymphocytes frombreast cancer patients was much greater than the range
from normal individuals (42% overlapped the ataxiatelangiectasia heterozygote range). It was proposed that inaddition to those mutated genes already identified withfamilial breast cancer, a number of other genes of lower
penetrance may also predispose to breast cancer. It wassuggested that these genes may be involved in theprocessing of DNA damage [H21, S27]. Family studies
showed that the lymphocytes of first-degree relatives ofsensitive individuals were significantly more sensitive to x
rays than expectation; segregation analysis of family
members (95 individuals in 20 families) suggested that asingle gene accounts for most of the variability, but that asecond rarer gene with an additive effect onradiosensitivity may also be present [R36].
135. It has also been found that a subset of patients with
common variable immune deficiency (CVID) showedenhanced sensitivity to G2 chromosomal damage [V1].
Among disorders with primaryimmune deficiency, CVID andataxia-telangiectasia have the highest reported incidence oftumours, and both disorders show similar types of tumours,suggesting a common risk factor. This finding may relate to
the link noted above (Section II.B.1) between immune systemdysfunction and radiosensitivity. However, it is clear that theG2 chromatid assay will detect enhanced levels of damage incells showing a normal response in survival assays (e.g.
xeroderma pigmentosum cells), and it may be argued that itis primarily a means of detecting genetic instability.
136. Another method based on alterations in the sedimentation of human lymphocyte nuclei is claimed to detect
particularly sensitive individuals [S4]. When the repair ofdamage is measured by return to normal sedimentation
properties after irradiation, it was possible todetect individuals
sensitive to radiotherapy and patients with autoimmune
disease [H5]. Further, patients showing post-therapycomplications were found to be more sensitive by this sedimentation
assay than those without complications [D2].
137. It has been suggested that individuals heterozygous forthe ataxia-telangiectasia defect are also at increased risk ofcancer. Swift et al. [S13] compared prospectively theincidence of cancer in 1,599 relatives of ataxia-telangiectasia
patients in 161 families in the United States and found anincreased relative risk of 3.8 in men and 3.5 in women for all
cancers, and an increased relative risk of 5.1 for breast cancer
in women. If borne out, this predisposition would contributesignificantlyto the cancer incidence in the general population:
heterozygotes are much more frequent than ataxiatelangiectasia homozygotes (both genes mutant) and fromthese data could constitute as much as 5%-8% of all adults
with cancer [S12]. However, commentary following the
publication of the work of Swift et al. [S13] questioned two
aspects of their findings. First, bias in the control group was
suggested [B24, K22, W14], although this was contested by
Swift et al. [S41]. Second, against a background radiationlevel of 1 mGy a-1, it was considered surprising that anincrease in breast cancer could be detected following
diagnostic x-ray procedures of #10 mGy [B24, L25].
Differences in dose rate might influence the effectiveness ofthese two sources, and in other studies an acute dose as low as
16 mGy gave a significant increase in breast cancer in normal
women aged 5-9 years at exposure [M26]. However, it is clearthat a relatively large difference in sensitivity of the ataxiatelangiectasia heterozygotes tothe acute diagnostic procedures
would be required to attribute the observed cancer incidence
to deleterious effects of one mutated copy of the ataxiatelangiectasia gene.
138. Genotyping of 99 ataxia-telangiectasia families using
markers tightly linked to the ATM gene showed that 25 of
33 women with breast cancer were heterozygous comparedwith an expectation of 15 of 33 (relative risk = 3.8; 95%
CI: 1.1!7.6) [A18]. Two smaller European studies ofcancer incidence among ataxia-telangiectasia heterozygotes
tend to support the data from the United States, giving an
overall relative risk for breast cancer of 3.9 (CI: 2.1!7.2)[E4]. The average relative risk for other cancers was lower
at 1.9, with the European studies showing no statisticallysignificant increase over controls. Using these combined
data, the proportion of breast cancer cases due to ataxiatelangiectasia carriers would be about 4%. This small
proportion is consistent with an inability in two studies[C36, W31] to detect linkage to AT gene markers in breast
cancer families (i.e. on the basis of the risk estimates andgene frequency, most cases of familial breast cancer will becaused by other genes) [E4].
139. The identification of the gene mutated in ataxiatelangiectasia (ATM, paragraph 56) has led to further
attempts to correlate this gene defect with breast cancer
incidence or with severity of response to radiotherapy. In a

ANNEX F: DNA REPAIR AND MUTAGENESIS

study of ATM mutations in 38 sporadic breast cancers, onlyrare polymorphisms were detected, none of which would leadto truncation of the gene (paragraph 57), giving no evidence
for an increased proportion of ataxia-telangiectasia heterozygotes in breast cancer patients [V8]. The same workers also
looked at 88 unrelated primary breast cancer cases fromfamilies previously associated with cancer susceptibility andfound three ATM mutations (3.4%); this number wasconsidered to be higher than expected by chance, but thesemutations did not necessarily segregate with cancer incidence
in the families [V9]. Screening a further 100 breast cancer
cases from families with a history of breast cancer, leukaemia,
and lymphoma revealed only one mutation, consistent withminimal involvement of the ATM gene [C65]. Fitzgerald et al.
[F21] detected heterozygous mutations in 2 of 202 (1%)
healthy women with no history of cancer, compared to 2 of
410 (0.5%) patients with early-onset breast cancer, consistent
with a lack of association. Similarly, in a study of 18 families
associated with a high incidence of breast and gastric cancers,
only one ATM mutation was found, and this did notcosegregate with the gastric cancer in the family [B58]. In a
study of 41 breast cancer patients showing marked normal
tissue response to radiotherapy, one was found with aheterozygous mutation in the ATM gene (2.4%), comparedwith none in a comparable number of control patients [S69].
The conclusion that mutation of ATM is not a major cause ofradiotherapy complications was supported by studies ofsmaller numbers (15-16) of breast cancer patients showing
severe normal tissue damage following radiotherapy, in whom
no ATM mutations typical of those in heterozygotes were
found [A22, R31]. In contrast, a relatively high frequency of
ATM mutations has been found for a group of prostate cancer
patients (3 of 17, 17.5%) with severe late responses toradiotherapy [H47]. The majority of these studies suggest thatheterozygosity for the ATM gene is not an important cause ofbreast cancer susceptibilityor severe response to radiotherapy,
but most do not have sufficient numbers of patients to exclude
completely a role for ataxia-telangiectasia heterozygotes.
140. The association of certain forms of lymphocytic
leukaemia with mutations in the ATM gene has alreadybeen noted (paragraph 59). Individuals with less severe
mutations in the ATM gene, allowing some expression of
ATM protein (paragraph 57), may also be at greater risk ofdeveloping breast cancer [S78]. It has also been observedthat loss of heterozygosity (Section IV.B) at chromosome
11q23 is a frequent occurrence in breast carcinomas [C27,H15]. At least two regions of 11q23 are commonly deleted,and one of these includes the ATM gene. Other common
human malignancies such as lung, cervical, colon, andovarian carcinomas, as well as neuroblastoma andmelanoma, also show an association with loss ofheterozygosity at 11q23 and may include the ATM generegion [R9]. In a recent study, 40% loss of heterozygosity
was found for markers of ATM in cases of sporadic
invasive ductal breast carcinoma; in the same tumours,
markers for the BRCA1 and BRCA2 genes showed 31%
and 23% loss of heterozygosity, respectively. Loss ofheterozygosity of ATM correlated with higher grade
tumours and a younger age at diagnosis [R32].

29

141. It is clearly important to detect individuals in thehuman population who are hypersensitive to radiation andto understand the connection between radiosensitivity andcancer-proneness. While there are a number of indicatorsof this hypersensitivity, it has been difficult on the basis ofcell survival assays to distinguish normal individuals fromthose carrying mutant genes giving intermediate levels of
sensitivity, when the full sensitivity range of normal cells
is taken into account (paragraph 128). In the case of
ataxia-telangiectasia heterozygotes, newer assays based on
G2 chromosomal sensitivity (paragraphs133!134), DNAdamage levels measured by the Comet assay [D22],
enhanced arrest in G1 [N2, N3], or enhanced arrest in G2[H6, L4, L5] all show more promise, but it is still not clear
whether any of the assays will detect these heterozygotes
exclusively. The discovery of the ATM gene (paragraph 56)
has made the task of detecting heterozygotes simpler, but
because of the large size of this gene it is still a difficult
task to screen large numbers of individuals for defects (seeparagraph 139). Since many other genes are known to
influence cellular radiosensitivity [Z13], it is likely that themolecular cloning of these genes will have a substantial
impact on the ability to determine the importance ofgenetic predisposition to human radiation risk. Indeed,
preliminary studies on nine recently-cloned repair genes
including XRCC1 and XRCC3 (paragraph 78) showed that
relatively common alterations in gene sequence exist in
normal individuals. An average of 14 percent polymorphic
sites yielding protein sequence variations was found insamples of 12-36 individuals, including some individuals
who were homozygous for the variant site [M53, S84].
While the functional significance of these variations has
yet to be established, their potential to reduce DNA repair
capacity may influence individual response to radiation
sensitivity and cancer susceptibility.

B. INFLUENCE OF REPAIR ON RADIATIONRESPONSES
142. It is known that DNA repair processes influence thesensitivity of mammalian cells and organisms to radiation.
If recovery from radiation damage is compromised, as incell lines defective in double-strand break repair, the slopeof the cellular survival curve is increased. Also, while
normal cells show a “shouldered” curve, the repairdefective cells commonly show a loss of this feature. In
simplistic terms, it is envisaged that repair processes innormal cells increase the chances of survival, but that as anacute dose increases, the amount of damage temporarily
saturates the repair capacity of the cell [G6]. There is very
good evidence in yeast cells that these aspects of survival
are associated primarily with the repair of DNA doublestrand breaks [F3]. It has been possible to make thiscorrelation in yeast, because DNA double-strand breaks
can be measured accurately, and mutant cells that aretemperature-sensitive for double-strand break repair areavailable. Thus, as the post-irradiation temperature isaltered, it is possible to see a concomitant alteration in
double-strand break repair and survival. Unfortunately, it

30

ANNEX F: DNA REPAIR AND MUTAGENESIS

has proved technically difficult to establish similar
quantitative correlations in mammalian cells, which havea much larger genome than yeast.
143. Operationally defined measures of cellular recovery,
termed potentially-lethal damage repair or sublethal damage
repair, have been used for some years in cellular "repair"studies. Potentially-lethal damage repair is measured by therecovery found when cells are held in a non-proliferative state
after irradiation, before respreading into fresh growth
medium. Sublethal damage repair defines the recovery seen
when survival is measured after splitting the dose over a
suitable interval. While it has not been clear whether
potentially-lethal damage repair and sublethal damage repair
measure the same underlying repair processes, it is notablethat both are absent in yeast single-gene mutants lacking
DNA double-strand break repair (rad52 series) [R2, R3]. It
has also been argued from kinetic data that double-strand
break repair is responsible for potentially-lethal damage repair
in yeast [F3]. Similarly, with low-dose-rate irradiation, thereis good evidence in yeast cells that DNA double-strand breakrepair is responsible for the dose sparing observed [F3].
Mammalian cell lines with defective double-strand breakrepair as well as ataxia-telangiectasia cells have also beenshown to lack potentially-lethal damage repair (xrs, XR-1,
ataxia-telangiectasia) or low-dose-rate sparing (xrs, ataxiatelangiectasia, irs2) (see Section II.A.2) [T30]. A correlation
has recently been found for human normal and ataxiatelangiectasia cells, irradiated at high or low dose rates,
between the amount of DNA double-strand breaks remaining
after repair and radiosensitivity (measured as cell survival)[F18].
144. Mammalian cells irradiated with densely ionizing
radiation, such as alpha particles, show both a more sensitive
response (increased slope of survival curves) and, commonly,
the loss of the curve shoulder region. Thus, characteristic
increases in RBE are found as the density of ionization (LET)
increases. This may well arise from the LET-dependence ofdamage complexity [A23, G28, G39] (see Section I.B), andrefined measurement of DNA breakage following bothx irradiation and high-LET particles has shown that there is
a comparative excess of small fragments for the high-LET
radiations [L53, R26]. However, several studies ofmammalian cells using conventional biochemical techniques
show that there is little or no difference in the numbers of
DNA double-strand breaks induced by low- and high-LET
radiations (RBE = -1) but that fewer of the breaks induced byhigh-LET radiations are repaired [B52, C53, J4, L52, P27,
P28, P37, R27]. Using these methods, a good correlation has
been found between the relative number of non-rejoined DNA
breaks and the RBE for mammalian cell inactivation over awide range of LET [G32, R25]. Plasmid DNA, irradiated togive the same amount of damage by gamma rays or by alphaparticles, showed much less repair of the alpha-particle
damage when exposed to mammalian cell extracts [H41]. It
is suggested that damage induced by high-LET radiations ismore complex, because of the increased local clustering ofionizations, and therefore less repairable than low-LET
radiation damage (see Section I.B). This finding is supported

by measurement of the rejoining kinetics of large fragments ofcellular DNA, where rejoining was generally slower after
irradiation with high-LET particles than with x rays [L62].
Additionally, using this method the proportion of mis-rejoined
fragments increased with x-ray dose (10%-50% over the dose
range 5-80 Gy), while similar experiments with high-LET
radiation gave a constant 50% of fragments mis-rejoined at all
doses tested [L72, K47].
145. In radiosensitive cells, specifically ataxia-telangiectasia
cells [C10] and the xrs series [T7], the increase in RBE with
LET is very much reduced. This result would be expected if
the damage caused by densely ionizing radiation is
intrinsically more difficult for normal cells to repair than thedamage caused by sparsely ionizing radiation. On this basis,radiosensitive cells show little alteration in effectiveness as the
LET increases, because they are already inefficient at
repairing low-LET radiation damage. This analysis suggeststhat the RBE-LET relationship is largelycaused bythe normal
cell's ability to repair low-LET radiation damage.
146. Extensive studies in bacteria have shown that some
repair processes are inducible by treatment with DNAdamaging agents [W11]. That is, DNA damage causes anincrease in the expression or activity of repair enzymes,
mitigating the effects of the damage. This process is part ofa much wider series of so-called stress responses by whichcells adapt to their environment. Knowledge of such
inducible processes in mammalian cells is fragmentary,
and in some cases the data are controversial. Evidence forthe existence of inducible repair processes in response toionizing radiation damage comes from several different
types of experiment. These may be broadly categorized as(a) adaptive response of pre-exposed cells (see also [U2]),(b) refined analysis of survival in low-dose regions, and (c)direct molecular evidence for the inducibility of specificgene products.
147. Pre-exposure to low doses of tritiated thymidine (1837 Bq ml-1) or x rays (5-10 mGy) was found to decrease thefrequency of chromosomal breaks in proliferating human
lymphocytes irradiated subsequently with a higher dose
(1.5 Gy) of x rays [O2, S34]. The aberration frequency is
reduced to about 60% of cells not receiving pre-exposure[S30]. Pre-exposure to low concentrations of radiomimetic
chemicals, such as hydrogen peroxide and bleomycin, canalso reduce the effect of a subsequent high dose of x rays
[C35, V4, W29]. This adaptive response is, however,stopped by 3-aminobenzamide, an inhibitor of poly(ADPribose) polymerase (paragraph 77), which is itself induced
in response to DNA breakage by radiation [S34, W23].
These features of the adaptive response have led to thesuggestion that low levels of DNA breakage act as a signal
for a response mechanism leading to accelerated repair ofradiation damage in mammalian cells.
148. Although increased resistance following low radiation
doses has been observed in a number of laboratories, it occurs
only in the lymphocytes of some individuals [B21, B26, H13,
S22, S26]. Also, lymphocytes from several persons with

ANNEX F: DNA REPAIR AND MUTAGENESIS

Down's syndrome [K16] and fibroblasts from homocystinuria
patients [Z11] did not show the adaptive response. The
adaptive response is usually found when lymphocytes areproliferating, not resting (G0) [S31, W15], although there aredata suggesting that pre-irradiation in G0, followed by achallenge dose in G2, gives increased resistance [C63, K16].
These observations have led to suggestions that the increased
resistance is a result of cell-cycle perturbation in the lymphocyte populations of some individuals following the preexposure (i.e. the cells sampled following the second dose arein a more resistant phase of the cycle than if no pre-dose had
been given). This idea has received some experimental
support [A9, W27], but recently an adaptive response forchromosomal and cell-killing responses has been found incells other than lymphocytes when irradiated in proliferating
or non-proliferating states [I1, L27, M24, S23]. Thus, thecauses of the adaptive response remain controversial, but
further work with different cell types has outlined the
responses affected and the conditions under which the
response is observed.

31151. The repair of DNA base damage is also a potentiallyrelevant process; a newly developed ultrasensitive assay forthymine glycols (paragraph 17) has shown that pre-exposure
to 0.25 Gy from gamma rays increases the repair of this typeof base damage from a subsequent dose of 2 Gy. The initialrate of removal of thymine glycols in human cellular DNA
was found to be increased by a factor of 2 following preexposure [L60].

149. Measuring chromatid breaks or chromosome-typeexchanges in lymphocytes, the maximum effect on resistanceto a second dose occurs within 5-6 h following the preexposure [S34, W15]; the phenomenon may persist for up to
three cell cycles [C24, S34]. While pre-exposure to relatively
high acute x-ray doses (e.g. >200 mGy) does not give theadaptive response [C46], when 500 mGy was given at low
dose rate (e.g. 10 mGy min-1) the adaptive response wasfound. Conversely, there appears to be a minimum dose rate
for the response induction by low doses (e.g. for 10 mGy predose, the dose rate must be >50 mGy min-1 for full effect)[S33]. A pretreatment with an acute dose of 20 mGy fromx rays will confer resistance to chromosome breakage by
150 mGy alpha particles from radon [W28]; however, whenacute high-LET radiation was used to give the pre-exposure,there was no increased resistance to a second dose [K16,W24].

152. New methods to measure with improved accuracy thesurvival response of mammalian cells have led to thediscoverythat the dose-response curve may initially be steeper
at low doses (<1 Gy) than predicted from the curve found at
higher doses. This response has been characterized as an
initial hypersensitivity at doses up to 0.5 Gy, followed byincreased radioresistance of the cell population [L23, M17,
W32]. The possibility that the hypersensitive response to low
doses is the result of differentially sensitive fractions in cell
populations has been rejected on the basis of two observations.
First, survival curves for radiosensitive tumour cell lines andan ataxia-telangiectasia line appear to show no changes insensitivity in the low-dose region. Preliminary data for two
further radiosensitive lines, one with reduced DNA doublestrand break repair and the other reduced excision repair, also
show no increased radioresistance at low doses in comparison
to their respective parental lines [S35]. Second, it is argued
that the hypersensitivityseen would require one fraction of thecell population to have an unreasonably high level ofsensitivity (e.g. about 7% of the cells to be >10 times moresensitive than ataxia-telangiectasia cells) [L24]. In parallel tothe adaptive response of lymphocytes, recent data also showthat pre-exposure to low doses from x rays or low concentrations of hydrogen peroxide increases radiation resistance inthe hypersensitive region. Further, the additional resistancefrom pre-exposure was transitory, requiring time for development and diminishing after two or three cell cycles [M18].
Again, it has been found that low-dose hypersensitivity isabsent with high-LET radiation [M17, M19].

150. Pre-exposure to low doses from x or gamma rays has
also been shown in several different cell types to decrease thefrequency of cell killing [L27, M24, S32], mutation [K13,
R12, S20, U17, Z8], apoptosis [F26], and morphologicaltransformation [A15, R33]. However, the last result is still
controversial. Using a near-diploid mouse skin cell line(m5S), Sasaki [S23] showed that x-ray-induced cell killingand chromosomal and mutation responses, but notmorphological transformation, are mitigated by acute preexposure to 20 mGy from x rays. A malignant derivative of
m5S cells lacked the adaptive response, but this was restored
along with morphological reversion bytransfer into these cellsof human chromosome 11. The study also showed thatchemicals that either activate or inhibit protein kinase C,
which has an important role in signal transduction, either
mimicked or abolished, respectively, the adaptive response[S23]. There have been reports suggesting that capacity torejoin radiation-induced DNA double-strand breaks is greater
following low-dose pre-exposure [I4, Z8], as well as data
implicating activation of antioxidant metabolism in theadaptive response {B65, Z14].

153. Multi-laboratory experiments attempting to measure
the dose response for chromosomal aberration induction in
lymphocytes from two normal donors at low x-ray doses(down to 4 mGy) also appear to identify departures fromfitted dose responses that have been attributed to the
induction of repair processes [P15]. It was then suggested
that the control value in these experiments was excessively
high, biasing the result towards non-linearity [E5]. However, analysis of lymphocytes from individuals in Austria
before and after exposure to fallout from the Chernobyl
accident, where a peak twofold increase in radiation
exposure was recorded, also showed dose-response curves
departing from linearity (i.e. decreasing or levelling off) at
annual doses between 0.3 and 0.5 mGy [P17]. Further invitro studies with x rays and larger numbers of donors havenot confirmed a significant departure from linearity at low
acute doses (down to 20 mGy), although statisticalvariation in the small numbers of aberrations detected atlower doses do not allow conclusive statements on dose
response [L37, L38]. These further studies did show an
excess of multiply-damaged cells in some donors after

32

ANNEX F: DNA REPAIR AND MUTAGENESIS

irradiation, leading to the possibility that a small subset oflymphocytes is especially sensitive to aberration induction byvery low doses. Similar studies with 15 MeV D-T neutrons
did not reveal significant departures from linearity at low
doses [P16]. Similarly, a study of chromosomal aberrationinduction in Syrian hamsters injected with 137Cs to give awhole-body dose of about 0.4 mGy, to mimic the Chernobyl
fallout exposure, failed to reveal a significant increase above
background values [L61]. Thus, the possibility that very low
doses give a higher yield of chromosomal aberrations than
expected on the basis of a linear extrapolation from high-dose
data remains contentious.
154. Reports suggesting that low doses of ionizing
radiation induce repair processes in vivo pre-date thecellular studies described above. Irradiation of mice withlow doses of x or gamma rays has produced evidence ofincreased resistance to subsequent higher doses to both
somatic and germ cells [C24, F6, W26]. Recent data showthat the marked cytogenetic adaptive response of mousegerm cells does not influence the response of the somaticor germ cells of the offspring [C25]. Examination ofchromosomal aberrations in the lymphocytes of people
exposed occupationally to higher-than-average doses ofalpha particles from radon (0.01-16 mGy) plus gamma rays
(1-3 mGy) showed that the dose-effect curve was not
related to that found at higher doses. Individuals subjectedto the lowest doses gave a steep increase in aberration
frequency with dose, while at higher doses the curve
flattened out [P14]. Tuschl et al. [T33] showed that thepeople exposed to the higher doses (8-16 mGy a-1) of alphaparticles gave higher levels of repair (unscheduled DNA
synthesis after UV light damage) in lymphocytes,
compared to controls. Hospital workers exposed to lowlevels of x and gamma rays (maximum annual dose =28 mSv) showed a reduction in the frequency ofchromosome aberrations induced by a dose of 2 Gy to theirblood lymphocytes, compared to non-exposed controls[B20]. However, it has been found that the lymphocytes ofchildren living in areas contaminated by the Chernobyl
accident showed no evidence of increased resistance tox rays [P9, U16], and separate reports suggests that fewer
people from the region of the accident showed the adaptive
response in lymphocytes compared with a control group[P19, M52].
155. A growing number of specific genes and proteins havebeen shown to be induced or repressed following irradiation,
mostly using relatively high doses from x or gamma rays (26 Gy). While these do not as yet form coherent pathway(s)coordinating response to radiation, they do implicate genes
and proteins involved in a variety of important molecular
processes (see also Section II.B). Induced proteins include
oncogenes/ transcription factors (c-jun, c-fos, interleukin-1,
and egr-1), proteins involved in cell-cycle regulation (p53 andcyclins A and B), growth factors, and DNA-metabolizing
proteins [PCNA, β-polymerase, and poly(ADP-ribose)polymerase], as well as the products of a number of unknown
genes [B25, K15, W20]. The development of methods for
rapidly screening hundreds or thousands of genes (micro-

arrays) in one experiment, for changes in levels of gene
expression, is beginning to show the extent of transcriptional
response to radiation damage. For example, using an array ofmore than 600 genes, gamma irradiation of a myeloid cell line
showed induction of 48 genes by factors of two or more, andmany of these genes had not been previously reported asradiation-inducible [A25]. Considering those genes which
respond differentially in the presence or absence of p53, it was
found using micro-arrays that several genes encoding secreted
proteins with growth inhibitory functions were upregulated ina p53-dependent fashion following gamma irradiation. Thus,
the p53 response to radiation may also be involved in growth
inhibitory effects on surrounding cells, as observed in“bystander” effects [K44]. Methods are being developed
similarly for assaying large numbers of proteins, to revealtranslational and post-translational responses, using 2dimensional gel systems coupled to mass spectrometry [B64,C69]. These methods will also revolutionize the classificationof normal tissue and disease states, in particular cancer, bypermitting a molecular description of the complete profile ofgene products present [G40]. Such classifications will be
invaluable in understanding the mechanistic basis of cancer
induction by agents such as ionizing radiation.
156. Increased or decreased levels of certain gene products
have also been found after low doses of radiation, within
the range inducing the adaptive response [B18]. Doses ofless than 500 mGy gamma rays have been found to induce
the expression of a variety of stress-response genes [P35,P36], and dose-responses of several of these genes wereshown to be approximately linear between 20 and 500 mGy
gamma rays [A26]. Some candidate gene products with apotential role in induced radiation resistance have been
identified, including a member of the heat shock protein 70
family (PBP74 [S79]), a heat-shock related immunophilinprotein (DIR1 [R37]), ribonucleotide reductase [S81], andthe MAPK and PKC protein kinases [S80].

C. SUMMARY
157. When the cells of normal individuals are examined forradiosensitivity, as shown by their survival in culture, variation by a factor of 2 is seen; this factor may be extended to 3
or 4 with the use of low-dose-rate irradiation conditions. It has
been suggested that this variation has a genetic basis, but it
has been technically difficult to establish this. Additionally, afraction of cancer therapy patients suffers from severe skinreactions, and cells from these patients commonly show aslightly elevated radiation sensitivity.
158. Assays based on chromosomal damage in G2 cells
have also been used to estimate radiation sensitivity innormal individuals and in breast cancer patients, relative
to individuals with known radiosensitivity disorders. The
response of a relatively large fraction of breast cancer
patients overlapped that of carriers of the ataxia-telangiectasia defect, suggesting that a number of genes involved in
response to radiation damage may predispose to this formof cancer.

ANNEX F: DNA REPAIR AND MUTAGENESIS

159. It has been suggested that individuals carrying one
defective copy of the ataxia-telangiectasia gene (ATM heterozygotes) are at increased risk from cancer, especially breast
cancer. A number of studies have recently tested this idea andcome to the conclusion that ATM heterozygosity is not an
important cause of breast cancer susceptibility (or severe
response toradiotherapy), but commonlytoo fewpatients havebeen tested to exclude completely a role for ATM. Specifictypes of tumours, such as T-cell prolymphocytic leukaemia,
are associated with high levels of mutation of the ATM gene(paragraph 59). Additionally, recent evidence suggests that thegenes involved in familial susceptibility to breast and ovariancancers (the BRCA genes, paragraph 72) are involved in DNA
repair processes and lead to radiation sensitivity when
defective in mice.
160. Repair processes affect the shape of survival curves,especially through differences in the processing of DNA
double-strand breaks, as seen in specific repair-defectivelines. The response to double-strand breaks probably also
underlies operationally-defined measures of cellular
recovery such as potentially-lethal damage repair andsublethal damage repair. High-LET radiations may not
induce a higher frequency of DNA double-strand breaks
than low-LET radiations, but high-LET radiation damage
is much less repairable. The increase in RBE with LET,
found for normal cells, is largely determined by the cell sability to repair low-LET damage.
7

161. Evidence for the inducibility of repair processes inmammalian cells is fragmentary. Data from pre-exposures

33

of cells to low radiation doses, as well as refined survival
analyses at low doses (<1 Gy), show an altered response
suggestive of an induction process but to date have failed
to link this response to known inducible processes.
However, recent experiments have shown that certain
genes in at least two repair pathways are up-regulated
following radiation damage (paragraphs 84 and 151).
Additionally, refined methods for looking at protein
structure and activity are beginning to reveal insights into
more subtle modifications following irradiation. Somedamage-response proteins may be activated by posttranslational modifications (e.g. phosphorylation, paragraph 102), without any change in the amounts of these
proteins, as part of a signalling mechanism promoting therepair of DNA damage. Other proteins have been shown toaccumulate in the cell to form discrete foci following
irradiation (paragraphs 71 and 74); these foci may
represent repair protein complexes accumulating at sites ofdamage. While there has been little exploration of the dose
dependence of these events, the data indicate thatmodification of cellular response mechanisms can occur
following irradiation, raising the possibility that dose
responses are altered as a result. Methods for dataacquisition on the inducibility of gene products arepresently being revolutionized by the introduction of
micro-array and complementary techniques, so that rapid
progress in this research area is to be expected. The
potential importance of inducible repair processes indetermining responses to low doses of ionizing radiation isconsidered further in Annex G, “Biological effects at low
radiation doses”.

IV. MECHANISMS OF RADIATION MUTAGENESIS
A. MUTATION AS A REPAIR-RELATEDRESPONSE
162. The cellular processing of radiation-induced damage to
DNA by enzymes may result in a return to normal sequenceand structure (correct repair). Alternatively, the processing
may fail or may cause alterations in DNA, with the consequence of lethality or inherited changes (mutations). It isalso possible that some subtle forms of damage may betolerated by the cell, particularly if it is non-replicating, andlead to persistent lesions in DNA. These lesions would haveto be both chemically stable and not be substrates for repair
enzymes and may include some minor types of damage suchas methylated bases generated by non-enzymatic alkylation[L68].
163. It is likely that simple base damage or loss in the
mammalian genome will commonly lead to base-pair
substitutions. In recent years the defined production of single
types of damage, at specific sites in DNA molecules in vitro,
has given insights into their consequences. For example, DNA
molecules carrying a single site of base loss (abasic site) havebeen shown to give rise to base-pair substitutions at that site,

either when introduced on shuttle vectors [G8] or when the
RAS gene was transfected and stable transformed clones
selected [K7]. These substitutions (a form of point mutation)
will often give rise to alterations in a gene product so that it
works less efficiently or not at all.
164. Breaks in DNA, especially double-strand breaks, arethought to lead to larger alterations such as deletions andrearrangements. Some alterations are very large and are seen
as chromosomal aberrations. While there is little formal proofof these relationships between breaks and mutation type, it has
been shown that the transfer of restriction endonucleases into
mammalian cells, causing site-specific DNA double-strand
breaks, gives rise to mutations and chromosomal aberrations[T24]. Additionally, the processing of isolated double-strand
breaks in defined DNA molecules by human cell extracts
show that these may lead to large deletions of surrounding
sequence [T10].
165. Reduction in the efficiency or fidelity of damage repair
may lead to an increase in genetic change. This is seen
strikingly in the recent discovery that loss of DNA mismatch
repair capacity is involved in specific forms of cancer.

34

ANNEX F: DNA REPAIR AND MUTAGENESIS

Mismatch repair is a form of base-excision repair (Section I.B)
that removes bases that have been altered or incorrectlyplaced(by a polymerase) so that the two strands of DNA do notmatch in base sequence. The cancer connection was first noted
as a high frequency of mutation of short-repeat-sequence(microsatellite) DNA in colon cancers, including hereditary
non-polyposis colon cancer. It was rapidly established that thegenes determining hereditary non-polyposis colon cancer colocalize with mismatch repair genes on human chromosomes
2 and 3 [B12, L15]. More genes with homology to thesemismatch repair genes have since been identified, and at least
two of these have also been shown to be mutated in the germline of hereditary non-polyposis colon cancer patients [N10].
Colorectal cancer is one of the most common human cancers
and perhaps the most frequent form of hereditary neoplasia;
hereditarynon-polyposis colon cancer accounts for as much as
5% of all cases of colon cancer, and the involvement ofseveral genes in the phenotype may account for its prevalence[M20].
166. The high frequency of repeat-sequence mutation wasrecognized as characteristic of loss of the mismatch repair
system (MutHLS), known for many years in bacteria andinvolving the concerted action of three mismatch enzymes tocorrect errors caused primarily during DNA replication (suchas slippage of strands at repeat sequences) [M27]. Patientswith hereditary non-polyposis colon cancer are therefore
thought to have a mutator phenotype similar to mutant strainsof bacteria with defects in mismatch repair. The role ofinactive mismatch repair genes in humans is expected to be
similar to the part played by tumour-suppressor genes; agerm-line mutation in one of the mismatch repair genes isfollowed by somatic mutation of the second gene copy during
tumour development [H22, L36]. However, the mismatch
repair genes are not thought to be directly involved in cancer;
rather, theyincrease genetic instabilityand the probabilitythat
random mutations will affect those genes critical to cancer
formation. This idea does not necessarilymean that mutations
alone are sufficient for cancer formation; it is interesting tonote that a subset of hereditary non-polyposis colon cancer
patients carry numerous mutations in their cells and the
expected defect in mismatch repair but have unexpectedly few
tumours [P10]. Given the proposed mechanism, it is curious
also that the increased cancer risk in hereditary non-polyposis
colon cancer patients is selective; while other sites areaffected, there is, for example, no increased risk for cancers of
the breast and lung [W17]. Recent success at breedingtransgenic mice defective in mismatch repair genes has shownthat theyare viable, but a large proportion develop lymphomasand sarcomas at an early age [B16, D9, R11]. While many ofthese mice die early, commonly succumbing to T-cell
lymphoma, those that survive for more than 6 months develop
gastrointestinal tumours, suggesting that these mice may be
used as a model for human colon cancer [H50].
167. Cell lines lacking mismatch repair also show anincrease in mutations at sites other than microsatellites. In
colorectal carcinoma lines, shown to have high frequencies of
microsatellite variation, the HPRT gene mutation rate was also
found to be increased by a factor of more than 100 over therate in normal human cells [B23, E8]. In one of these lines,
known to have a defective human MLH1 mismatch repair
gene, about one quarter of the mutants had point mutations(frameshifts) at a hotspot within a run of guanine bases in the
HPRT gene [B23]. An extensive study of one mismatch
repair-defective line using the APRT gene similarlyshowed anelevation of frameshift mutations at sites of repeat baseTA transversions at sites ofsequence, as well as AT
secondaryDNA structure [H23]. A potentiallyrelevant site formutations of this type is in the BAX gene, involved inpromoting apoptosis (Section II.B.3); more than half of 41colon carcinomas with microsatellite instability also hadframeshift mutations in a run of eight guanines in the BAX
gene [R19]. These findings are consistent with a role formismatch repair in correcting base misalignments generated
during DNA replication in normal cells. Interestingly, lack ofmismatch repair also allows cells to become tolerant of certain
forms of induced DNA damage. Alkylation of DNA bases
normally produces cell-cycle arrest and/or death and can leadto cancer, but mismatch-repair-defective cell lines are highly
resistant to the effects of alkyating agents [B28, K12].
Mismatch-repair-deficient mammalian cell lines also show asmall but significant increase in resistance to gamma rays
[F19], suggesting that repair of certain types of radiationinduced base damage is recognized by the mismatch repair
system (e.g. 8-oxoguanine, which mispairs with adenine,
paragraph 24). Mouse embryonic stem cells, heterozygous fora defect in the mismatch repair gene Msh2, showed resistanceto low-dose-rate gamma radiation (0.004 Gy min-1) but not toacute radiation (1 Gy min-1). On the basis of this result, it was
speculated that heterozygosity for mismatch repair may also
contribute to tumorigenesis in a direct manner, without loss ofthe other gene copy [D20]. The mechanism of resistance isthought to follow from a reduction in abortive “repair”:
mismatch repair normally removes a base that is incorrectly
incorporated opposite a damaged base but this repair isabortive, because the damaged base remains in place and maylead to lethality.

÷

168. It is to be expected that the genes involved in otherfunctions required for the maintenance of genome stability
would lead to a similar effect of increasing mutation
frequency, thereby affecting cancer rates. These functions
would include the "proofreading" of DNA synthesis by polymerases and the regulation of DNA precursor synthesis.
Indeed, it has been shown that mutations in the exonucleasedomains of the DNA polymerase δ gene correlate with highmutation rates in some colorectal carcinoma cell lines lacking
changes in mismatch repair genes [D6].
169. Repair enzymes may cause mutations by virtue of theirimperfect response to damaged DNA, but in the last few years
it has become apparent that many organisms have also
retained specific enzymes that introduce mutations into
damaged DNA. Thus the loss of certain repair functions canbe antimutagenic, and indeed mutant strains of bacteria
showing no increase in mutant frequency after treatment with
DNA-damaging agents have been known for many years[W11]. Recent studies have defined these mutagenic or errorprone DNA repair processes in bacteria and yeast, and have

ANNEX F: DNA REPAIR AND MUTAGENESIS

B. THE SPECTRUM OF
RADIATION-INDUCED MUTATIONS
170. Ionizing radiation can induce many types of mutation,from small point changes to very large alterations
encompassing many genes. From recent studies ofmammalian cells, in which a few genes have been examinedin some detail, it is clear that radiation is most effective at
inducing large genetic changes (large deletions andrearrangements). This mutation spectrum (proportion ofdifferent types of mutations) differs from that foundspontaneously or the spectra induced by many other DNAdamaging agents (e.g. ultraviolet light, alkylating chemicals).
These other agents tend to induce mostly point mutations,
independent of the genomic region assessed. However, it has
also been found that the proportion of radiation-induced large
genetic changes can vary with the genomic region assessed.

usually be lethal [E3, T16, T21]. Thus, although the initial
occurrence of radiation-induced genetic changes may besimilar at different sites in the genome, lethality will limit
both the frequency and the apparent size of mutations
recovered. Mutations will be recovered in some genes at low
frequency, because they or the region they reside in cantolerate little change (certain genes have been found that,within experimental limits, show almost no radiation-induced
mutation [T15]), while mutations in other genes may be
detected at relatively high frequency because of cellular
tolerance to large changes. Knowing this, it is possible todevise mutation detection systems that tolerate very largechanges that would be inconsistent with survival in normal
diploid cells. The most extreme example of high-frequency
mutation detection is in a system devised by Waldren et al.
[W12] that uses as the mutational target an accessory humanchromosome 11 introduced into a Chinese hamster cell line.
Since no genes on the human chromosome are essential forcell survival, verylarge changes including whole chromosome
loss are tolerated, and mutant frequencies are about 100 times
greater than for endogenous genes such as HPRT.
172. Much of the mutagenic response to ionizing radiation
has been measured, for experimental ease, in regions of the
genome that are present at the level of one copy per cell.
This situation (monosomy) occurs naturally in parts of the
genome such as the X and Y chromosomes in males; also,functional monosomyappears to be quite extensive throughmechanisms that switch off gene expression in one copy
(as in one copy of the X chromosome in females and in
imprinted chromosomal regions). However, mostchromosomes and genes are present as two copies per cell(disomic; see paragraph 7). Where the loss of function in
one copy of a disomic gene is tolerated without harm (e.g.
because of a recessive point mutation), it has been foundthat the other gene copy is mutated at relatively highfrequency by ionizing radiation. Since the two gene copies
differ in their functionality (that is, they are heterozygous),
this mutation process is commonly termed loss of hetero60

APRT mutants in heterozygous cells
APRT mutants in hemizygous cells
HPRT mutants in hemizygous cells

50
MUTANTS PER 106 CELLS

revealed that similar processes occur in other organisms,
including humans [J23]. It has been found that genes coding
for special types of DNA polymerases are responsible formany of the small mutations (base substitutions, frameshifts)
occurring spontaneously and after treatment with DNAdamaging agents. In bacteria, for example, when DNA
damage blocks the normal replication process, an “SOS”
response is activated and more than 20 genes are induced[S83]. Among these genes are polymerases (e.g., DNA
polymerase V) with a high affinity for damaged DNA, which
are able to continue to synthesise DNA for a few bases in thepresence of damage (translesion synthesis) and which
commonly put in incorrect bases to give a mutation [R35,
T45]. Similarly, in yeast, three genes (REV1, REV3, REV7)
are required for much DNA-damage-induced mutagenesis,
and these have been found to specify mutagenic DNA
polymerase activity required for translesion synthesis [N23,N24]. A human gene named hREV3 has recently been cloned
through its homology to the yeast REV3 gene and been foundto have the properties of a mutagenic polymerase [G41]. As
well as this error-prone activity, yeast cells have anotherspecialized DNA polymerase (encoded by the RAD30 gene)that can perform translesion synthesis in an error-free way.
Strikingly, a human gene homologous to RAD30 (hRAD30)
has been found mutated in a variant form of the humansunlight-sensitivity disorder xeroderma pigmentosum (seeparagraph 26). Since individuals with this disorder suffer ahigh frequency of skin cancer, this result suggests that errorfree translesion synthesis is important to protect against
sunlight-induced cancers [J24, M49]. While much remains tobe learned about the operation of these specializedpolymerases, it seems clear that maintaining a balance
between error-free and error-prone pathways of translesion
synthesis is important in the determination of rates of (point)mutation in cells.

40

30

20

10

0
0

171. The size of genetic changes in a given region of the
genome is limited by the extent to which that region cantolerate change. The region may contain genes that are
essential for the viability of the cell and organism; if anessential gene is altered or lost, the changes incurred will

35

1

2
DOSE (Gy)34

Figure V. Effect of gene location (for hemizygous
genes) and copy number on mutant frequency inhamster cell lines carrying two copies of the gene
(heterozygous) or single copy (hemizygous) [B11].

36

ANNEX F: DNA REPAIR AND MUTAGENESIS

zygosity. The frequency of mutation in this disomic
heterozygous situation has been shown experimentally tobe higher than for the same gene in a monosomic situation[E3, M14, Y2], as illustrated in Figure V for the APRT
gene, where the mutation frequency difference is about 20fold [B11]. The reason for this higher mutation frequency
in the disomic situation appears again to relate to toleranceof the frequent large genetic changes caused by radiation.
Hemizygous
chromosome

Thus, the first copy, despite carrying a point mutation, still
has the remainder of that genomic region intact (including
any linked essential genes), and large deletions in thesecond copy can be tolerated because these do not lead to
a complete loss of linked essential genes. In a monosomic
chromosomal region, however, there is no other copy (orno functioning copy) present, and the mutant cell will notsurvive large genetic changes (Figure VI).
Heterozygous
chromosome pair

Chromosomal
regionGenes
in regionA(essential)

A
A

Point mutation

X
X

X

Target gene
(inessential)

B
B(inessential)

B

Radiation-induced large deletion(including gene X and parts of genes A and B)

Mutant survives

Mutant does
not survive

Figure VI. Consequences of radiation-induced deletion mutation in a hemizygous (monosomic) andheterozygous (disomic) gene.
Loss of function in gene X is selected for, but the mutation also removes parts of adjacent genes, one of which isessential for cell viability. The mutation results in death of the cell carrying the mutation when the genetic region ishemizygous, but not when it is present in a heterozygous state.

173. Where disomic genes are used to study radiationinduced mutation, the mutation frequency will also varydepending on which of the two copies (alleles) is used as thetarget. It is to be expected that in addition to the target gene,other linked genes will vary in their functional state(heterozygosity). Thus, if an active allele of the target gene islinked on one chromosome to the active allele of an essential
gene, deletions of that chromosome will be severely limited.
Conversely, linkage of the target gene to an inactive copy ofthe essential gene will not be limiting if the homologous

chromosome carries the active essential gene and will allow
the cell to survive a large deletion. Thus, a 10-fold differencein mutant frequency has been recorded for the two alleles ofa heterozygous TK gene in lymphoblastoid cells [A11]. Thisdifference in mutant frequency was associated with a class ofslow-growth mutants carrying large genetic changes at the siteof the mutable allele of the TK gene [A12].
174. High mutation frequencies may therefore mainlyreflect tolerance to large genetic changes, although higher

ANNEX F: DNA REPAIR AND MUTAGENESIS

frequencies may also arise from additional mutation
mechanisms available in heterozygous cells (paragraph 183).
Since ionizing radiation is relatively good at inducing large
genetic changes, it will be very effective as a mutagen for
disomic genes compared with its effectiveness for monosomic
genes (also, in general, ionizing radiation is less effective thanpotent chemical mutagens when used to mutate monosomic
genes). It may be that, for example, an individual inherits apoint mutation in one copy of a tumour-suppressor gene,which in itself does not lead to deleterious effects because ofthe "good" copy still present. However, should this person be
subjected to an agent like radiation, which is very effective at
inducing large genetic changes, the other gene copy can bereadily mutated along with much of the surrounding
chromosomal region, because that region is shielded by thepresence of the other gene copies. In this way radiation can beseen as a very effective mutagen for specific types ofmutational change; indeed it could be argued that forrecessive gene mutation, individuals are susceptible to agents
such as radiation if they already carry point mutations in
disomic genes. This phenomenon is an aspect of predisposition of individuals to specific genetic changes and should not
be confused with radiosensitivity (paragraph 122).
175. The prediction that large genetic changes would befound in disomic target genes has been borne out in studies ofmice [C19]. Screening progeny after x irradiation of malegerm cells revealed that very large chromosomal changes(deletions, rearrangements, and complex changes) occur athigh frequency in one copy of disomic regions of the genome;
these large changes were consistent with reasonable viability
and fertility in the progeny. As expected, when these mutant
regions are not shielded by a second normal copy of the genes
involved, they are invariably lethal to the mice.

37

C. MOLECULAR ANALYSIS OF
RADIATION-INDUCED MUTATIONS
176. While it can be argued that ionizing radiation has aparticular mutation spectrum, it has not been found thatany one type of mutation is induced specifically by ionizing
radiation. However, detailed analysis of mutations (and tosome extent of chromosomal aberrations) has given somepossible indicators of differences between spontaneouslyoccurring and radiation-induced mutations, and of differences between densely and sparsely ionizing radiation.
177. Sequence analysis of x- or gamma-ray-induced point
mutations in mammalian cells has shown that a variety oftypes occur, from base-pair substitutions and frameshifts tosmall deletions. Analysis of substitutions in the HPRT and
APRT genes has shown that all of the 6 possible types occur,
although at different frequencies (Table 3) [G10, M12, N11].
A majority of the radiation-induced substitutions weretransversions (alteration of the base from a purine to apyrimidine or vice versa), while many spontaneouslyoccurring substitutions were G:C A:T transitions. A larger
proportion of frameshift mutations also occurred in theradiation-induced mutants than spontaneously, and a few
more of the radiation-induced point mutations were multiple
substitutions (more than one base change in close proximity).
Using a small gene target transferred into mouse cells, little
difference was found in the mutation spectra for spontaeously-occurring and x-ray-induced mutations [K18]. The
frequency of small rearrangements and deletions was increased in both spontaneous and radiation-induced mutants,
indicating differences in the balance of mutagenic
mechanisms at the site of integration of the transgene
compared with those at endogenous genes.

ÿ

Table 3Comparison of spontaneous and radiation-induced point mutation spectra
APRT gene / hamster CHO cells [M12]

HPRT gene / human TK6 cells [N11]Type of mutation
Spontaneous

Induced by gamma rays
(2.54 Gy)

Spontaneous

Induced by x rays
(2 Gy)

Base substitution
Transition
Transversion

55 (71%)31 (40%)24 (31%)19 (66%) a
6 (21%)
13 (45%)10 (55%)6 (33%)
4 (22%)

19 (54%)7 (20%)
12 (34%) a

Frameshift

6 (8%)

5 (17%)

3 (17%)

8 (23%)

Small deletion or rearrangement

16 (21%)5 (17%)

5 (28%)

8 (23%)

77

29

18

35

Total
a

Includes two tandem substitutions.

178. Radiation-induced point mutations were found atsites widely distributed within the gene, while spontaneously-occurring point mutations tended to cluster at certain
sites [G10, M12, N11]. Differences in the types of basedamage responsible and in the randomness of damage
induction are likely to explain these differences.

179. Smaller numbers of large genetic changes have beensequenced because of the difficulty of locating and cloning the
breakpoints of large deletions and rearrangements. No new or
specific mechanism has been found for the induction of theselarger changes. In the hamster APRT gene, present in thehemizygous state, the large gamma-ray-induced deletions tend

38

ANNEX F: DNA REPAIR AND MUTAGENESIS

to have short direct or inverted repeat sequences around theirbreakpoints and to fall into regions rich in adenine-thymine
base pairs. Insertions, however, involved repetitive sequences
and were accompanied by short deletions [M13]. It wasnotable in these studies that large changes extending
downstream of the APRT gene are not found, suggesting thatan essential gene is present in this region.
180. In the human HPRT gene, also in the hemizygous state,large deletions are found to extend both upstream anddownstream of the gene over a region of about 2 Mb [M16,
N9]. Radiation-induced large genetic changes tend toeliminate the whole HPRT gene, making their analysis
difficult. Therefore a series of flanking markers has been
identified to delimit these changes and ultimately to map the
positions of the breakpoints. Sequence analysis of a few
deletion mutations has again shown the presence of short
direct repeats at some breakpoints induced by both x rays andalpha particles, as well as other sequence features in adjacentregions [M15, S18]. The large deletions induced by radiation
frequently include sequences adjacent to HPRT, and it has
been suggested that this may be a signature of radiationinduced events as opposed to spontaneous events [N17]. This
type of mutation was equally common with gamma-ray dosesof 0.2 or 2 Gy d-1. Use of methods amplifying specific regionsof the genome to measure rapidly the frequency of rearrangements following x- or gamma-irradiation, followed bysequencing the breakpoints, has given little indication of theinvolvement of specific sequence features or clustering of
breakpoints [F22, F23].
181. Shuttle vector systems consist of small defined DNA
molecules that may be mutated in mammalian cells but
rescued into bacteria for rapid analysis of sequencechanges. While these facilitate the molecular analysis ofinduced mutations, they generally have the drawback thatthe target genes are very small and are flanked by essential
sequence. This means that only point mutations aredetectable, and the major class of radiation-induced
mutations (large deletions and rearrangements) cannot beanalysed. However, Lutze and Winegar [L19] devised alarge shuttle vector based on Epstein-Barr viral sequencethat could be maintained episomally in human
lymphoblastoid cells and could detect changes of up to 8 kb
or so. Using this vector, the mutation spectra of both x rays
(150-600 Gy) and alpha particles (3 Gy) from radon gas
were studied [L19, L20]. A larger proportion of deletions
was found among alpha-particle mutations (64%) thanamong x-ray mutations (13%), but in a subsequent study
with lower doses of x rays, more deletions were found(41% at 100 Gy, 33% at 20 Gy) [L21]. For both x rays andalpha particles, these deletions were large (>2.4 kb), andtheir breakpoints were clustered in specific regions of the
shuttle vector. The breakpoints were commonly associatedwith short direct sequence repeats of up to six base pairs.
Use of the same vector system in a lymphoblastoid cell line
defective in double-strand-break repair gave a slightly
higher frequency of deletions after x irradiation, in
association with an additional class of small deletion
mutations (<2.4 kb) [L21].

182. The presence of sequence features such as short direct
repeats of a few base pairs at large deletion junctions suggeststhat illegitimate recombination (see Section I.B) has driven
the mutation process. Recently it has been possible to reconstruct the process of illegitimate recombination in cell-free
conditions to show that the process can be associated with a
DNA double-strand break. DNA molecules are broken at a
specific site, using an endonuclease, and exposed to extracts
from human cells for a brief period. Analysis of the extracttreated DNA shows that while the majority of broken
molecules are correctly rejoined, a small fraction (about 0.5%)
are mis-rejoined to give a deletion. The mis-rejoin mechanism
involves the pairing of short direct sequence repeats, situatedeither side of the break, so that the intervening sequence(including one of the repeats) is deleted [T10]. This mechanism has been found in both radiation-induced mutations andin germinal and somatic-cell mutations in humans. A modelfor this process is shown in Figure IV. In addition to simple
deletion, this cell-free system detected a small fraction of more
complex changes, e.g. a large deletion associated with theinsertion of several hundred base pairs.
183. In heterozygotes, in addition to the tolerance of largechanges, additional mechanisms of mutation may occur.
The two gene copies may undergo mitotic recombination
or non-disjunction, leading to the appearance of mutations.
However, where loss of heterozygosity is measured simply
by the presence or absence of the active (wild-type) gene,
it is difficult to distinguish mutations occurring by these
additional mechanisms from large deletions. In a study ofmutation in the human TK gene, using both linked marker
analysis and densitometry to assess gene copy number,about 50% of the spontaneous mutants appeared to involve
recombination, while x-ray-induced mutants were mainly
deletions [L29]. Recombination was similarly found to be
involved in spontaneous mutation of the APRT gene inhuman cells, but this mechanism was also detected in a
number of gamma-ray-induced mutants [F8].
184. While it is difficult to compare somatic-cell mutation
data with animal germ-cell data, some recent molecularanalyses of the genes used in mouse specific-locus tests
indicate possible similarities in the types and mechanismsof mutation. For example, with some dependence on bothcell stage and radiation quality, a large proportion ofalbino (c) locus mutations on mouse chromosome 7
recovered from germ-cell irradiations are large deletions[R14]. Molecular analysis of radiation-induced mutations
at the c locus shows that a genomic region of 1.5-2 Mbaround the tyrosinase gene may be deleted in mutants
without leading to inviability in homozygotes [R13]. Thistarget size is very similar to that of the human HPRT generegion used in somatic-cell mutation studies. Also,
mutation frequencies per unit dose of low-LET radiation at
the c locus in mouse germ cells and at the HPRT locus inhuman somatic cells are in order-of-magnitude agreement[T19], given that mutations at the c locus occur with about
average induced frequency relative to other loci in specificlocus tests [S29]. Comparisons of this type emphasize thatthe mechanisms of radiation mutagenesis are similar inANNEX F: DNA REPAIR AND MUTAGENESIS

185. Comparison of two human lymphoblastoid cell lines
derived from the same original cell line has given further
insights into the mechanisms of radiation mutagenesis.
Line TK6 is more sensitive to the lethal effects of x rays
than line WIL2-NS, but the converse is true for mutation
response to x rays (although not to chemical mutagens).
The increased frequency in WIL2-NS at the hemizygous
HPRT gene was modest (factor of 4 at 2 Gy), while theincrease for the heterozygous TK gene is by a factor of 2050, depending on the TK allele used as the target [A13].
Molecular studies have revealed that all TK mutants in
WIL2-NS arose by loss of the active allele and linked
markers (over a 5 Mb region), while point mutations andless extensive deletions were common in the TK6 line.
Two copies of the TK gene were present on a majority ofkaryotypes in the WIL2-NS mutants, as expected if thesearose from mitotic recombination rather than deletion,
while the converse was true for TK6 mutants [X1]. It
seems likely, therefore, that the WIL2-NS line has a higher
frequency of recombination, leading to a greater ability tosurvive x-ray damage but incurring a concomitant increasein mutation. Analysis of the p53 status (see paragraph 112)of these two cell lines has shown that p53 protein levels are
four times higher in WIL2-NS than in TK6, because of amutation in exon 7 of the TP53 gene in WIL2-NS (TK6 iswild-type) and that apoptotic death is substantially delayed
in WIL2-NS [X2, Z7].
186. In a study of B-cell precursors from Tp53-knockout
mice, it was also found that high frequencies of HPRT
mutations occurred following x irradiation but that theseresulted from a preferential survival of mutant clones
rather than from a p53-dependent increase in mutation rate
[G26]. It was concluded that loss of p53 function allows
mutated cells to survive that would otherwise be eliminatedby apoptosis. Other recent studies with cells from p53deficient mice have similarly concluded that they do nothave an intrinsically higher-than-average mutation rate
[C68, N12, S21].
187. It has been suggested that a relatively unique featureof radiation mutagenesis is the induction of complex
genetic changes [M11]. For example, a radiation-induced
deletion may be associated with a rearrangement at thesame site, and the rearrangement may be an exchange,
inversion, or insertion. Additionally, complex changes may
be seen as chromosomal exchanges involving several sites
in the genome following a single acute radiation treatment[S15]. However, because of their large size and complexity,
these changes have been difficult to analyse in cellular
genes at the molecular level. Further molecular analyses
will be necessary to establish whether or not large
mutations and chromosomal aberrations are formed bysimilar mechanisms. It is clear from studies with one or
two genetic regions, and especially the region containing
the human HPRT gene (Xq26), that genetic changes

identified at the molecular level as mutations can extend tosizes that are visible in the light microscope [S17].
However, the breakpoints of such very large mutations andchromosomal aberrations have not been sequenced.

D. EFFECT OF RADIATION QUALITY
188. In general, high-LET radiation induces a higher
frequency of mutants in rodent and human cells, per unit
dose, than low-LET radiation [B17, B19, C22, C30, F7,
G18, H20, L31, M22, M25, S88, S89, T16, T18, T32,
W22]. The RBE varies with LET, peaking at 100-200
keV µm-1, with values as high as 7-10 found for alphaparticles and heavy ions in this LET range [C22, T16]. In
some mouse cell lines, a relatively small RBE of 2-3 hasbeen found for HPRT mutant frequency with alphaparticles in the peak range, and when cell survival is taken
into account, there is no difference in the effectiveness ofalpha particles and x rays. This result appears to be due tothe very high effectiveness of low-LET radiation on thesecells, as reflected in increased cell killing and mutagenesis
for a given dose compared with other rodent lines [B19,I2]. That is, the mutagenic effectiveness of low-LET
radiation is more variable than that of high-LET radiation,
presumably because cell lines have different abilities torepair low-LET radiation damage (while high-LET
damage is less repairable; see Section III.B). An exampleof this variability is shown in Figure VII [T28].
30
EAT cells exposed tox rays and alpha particles
25
MUTANTS PER 10 5 CELLS

somatic and germ cells, and that if measurements are madeunder similar conditions of mutation tolerance, then themutation frequencies will be comparable.

39

20

15
V79 cells exposed
to alpha particles
10

V79 cells
exposed to x rays

5

0
1

0.5

0.2

0.1

0.05

0.02

SURVIVING FRACTION

Figure VII. Fitted curves of mutant frequencies in
Chinese hamster V79 cells [T18] and mouse ascites(EAT) cells [I20] induced by x rays and 238Pu alphaparticles (see paragraph 172).

189. A study of 19 non-smoking people living in houses
with a range of radon concentrations (21-244 Bq m-3 in the
United Kingdom suggested that there was a correlation of
HPRT mutant frequency in blood T-lymphocytes withradon concentration. These data suggested that alphaparticles in this LET range have a considerably higher
effectiveness than found previously in high-dose experiments with human somatic cells. While the estimation of

40

ANNEX F: DNA REPAIR AND MUTAGENESIS

alpha-particle dose to circulating blood lymphocytes isdifficult, the authors estimated an annual doubling dose of
2.1 mSv from their results [B29]. A number of problems
attend such measurements, not the least of which is the
sample size. In a follow-up study by the same authors witha total of 65 persons from 41 houses in the same town, no
significant correlation was found between mutant
frequency and radon levels, even for people living in thesame house [C34]. In a study in Belgium of 24 people
living in houses with radon concentrations exceeding 100
Bq m-3, a negative association between radon concentrationand HPRT mutation frequency was found [A10]. This
result led the authors to suggest the induction of repair
processes by low levels of radon.
190. The TP53 tumour-suppressor gene is commonly
mutated in many different types of cancers, with afrequency ranging from <10% to >50% depending on
tumour type. Analysis of mutations in the TP53 gene inlung tumours from uranium miners initially gave somehope that these would provide a means of fingerprinting
high-LET radiation damage. Vahakangas et al. [V2]
sequenced exons 5-9 of the TP53 gene in 19 lung tumoursfrom miners working in New Mexico. They found several
point mutations, although these lacked a class of basesubstitution (G:C T:A transversions) and were not at the
hotspots described for lung cancer. In contrast, in a larger
study of 52 lung tumours from Colorado uranium miners
exposed to a dose that was, on average, five times higher,
Taylor et al. [T12] sequenced the same exons and foundthat about half of the substitutions were of the G:C T:A
type in codon 249 (codon = base-pair triplet encoding one
subunit of the p53 protein). This transversion is very rareamong TP53 mutations in lung tumours (many of which
are presumed to be smoking-associated) and was suggested
to be a potential marker for radon-associated lung cancer.
At present, apart from the difference in average dose, it isdifficult to see why these two TP53 mutation studies gavesuch contradictory results. One possibility is that thetransversion mutations seen are a result of some otheragent in the environment of the mine; if the mine is damp,
then fungal toxins similar to aflatoxin B1, known to induce
this type of mutation, might be present [V3]. Follow-up
experiments looking specifically for the codon 249
transversion in lung cancers from individuals exposed tohigh domestic radon levels in the United Kingdom [L48]and from German uranium miners [B45] failed to find any
examples of this mutation.

ÿÿ191. In a study of thymic lymphomas in RF/J miceinduced by gamma rays or by neutrons (0.44 MeV), a
similar frequency of lymphomas was associated with
mutations activating RAS oncogenes (24% for gamma rays,
17% for neutrons). Of the gamma-ray set, 89% were
K-RAS-activated, with the majority (7 of 8) having a GAT
GGT mutation in codon 12. However, in the neutron set
no particular mutation site predominated, and one
lymphoma contained a K-RAS gene activated by a point
mutation in codon 146, a site not previously associatedwith any human or animal tumour [S36].

ÿ

192. There are conflicting data on possible differences in the
spectrum of large genetic changes induced by high-LET
radiation relative to those induced by low-LET radiation.
Early cytogenetic studies with diploid human fibroblasts
suggested that a greater proportion of HPRT mutants inducedby high-LET radiation carried large genetic changes than did
x-ray-induced mutants [C37, T19]. Studies of specific-locus
mutations in mouse spermatogonial stem cells had also
concluded, based on a number of criteria, that 239Pu-induced
mutants carried more severe genetic damage than mutants
induced by low-LET radiation [R16]. However, classificationof HPRT mutations by molecular analysis in both diploid
human cells and hamster cells has not shown differences in
the proportions of large deletions to point mutations at dosesof low- or high-LET radiation giving about 20% cell survival[A8, G16, S18, T20]. A confounding factor (commonly
underestimated) in determining mutation spectra can be thenon-independence of mutants when grown in bulk culture
after irradiation [T22], but in the most recent studies [A8,S18] the experimental design ensured the independence of all
mutants. Other studies have reported differences: irradiation
with 190 keV µm-1 Fe ions gave 82% large deletions of the
HPRT gene in human TK6 cells relative to 54% for x rays
[K21]. High doses of radiation from incorporated 125I, giving
1% survival of human TK6 cells, were reported to increase thefrequencyof deletions or rearrangements when compared withlower doses of 125I or with x rays [W22]. It was noted thatthese high-dose mutations commonly showed loss of part of
the HPRT gene rather than the entire gene. It has similarly
been reported in recent studies with the same cell line that the
average size of radon-induced deletions of the HPRT gene is
not as great as that induced by x rays [B19, C29]. This feature
has also been noted for mutations induced at the hemizygous
DHFR gene in hamster cells; that is, the frequency of totalgene deletions was much higher for gamma-ray- than foralpha-particle-induced mutations, while the reverse was true
for intragenic deletions [J11]. In the latter study, it was also
found that some alpha-particle-induced deletions sharedcommon breakpoint sites, indicating a non-random distribution. Use of the heterozygous TK gene as a target, also in TK6cells, showed again that the proportion of deletions was
similar for x rays and high-LET radiation (4.2 MeV neutrons
and argon ions), but that high-LET radiation induced acategory of large rearrangements not found for x rays [K20].
It has also been claimed that high LET radiations induce noncontiguous deletions that are not found with x or gamma rays(see also paragraph 208) [S90]. A number of these studies,
therefore, suggest there could be differences in the types oflarge genetic changes induced by high-LET as opposed to
low-LET radiation. Much more careful analysis is required
before any general statement can be made about the extent or
nature of these possible differences.
193. The mutagenic effects of a single alpha-particle
traversal were measured with a high-frequency detection
system (paragraph 171). Using a microbeam to localize 90
keV µm-1 alpha particles to cell nuclei, a dose-dependent
increase in mutant frequency was found for traversals of 1-8
particles, with a twofold increase for 1 alpha particle traversal(cell surviving fraction = 0.8) [H42]. Molecular analysis of

ANNEX F: DNA REPAIR AND MUTAGENESIS

these mutants gave some support for the view that higher
doses of high-LET radiation induce larger genetic changes,
but this system is unusual in being able to sustain loss of thewhole target chromosome without lethality.
194. It has been proposed that the ratio of specific forms ofchromosomal aberration may be a fingerprint of exposure tohigh-LET radiation [B13, S71]. A low ratio (about 6) ofinterchromosomal to intrachromosomal exchanges was foundin several studies of aberration induction by high-LET
radiation, while a value of $15 was found for x or gamma
rays. However it was subsequently demonstrated [S54], using
a simple two-dimensional chromosome model allowing theprediction of exchange sites relative to track structure, that
more densely clustered tracks are not predicted to give asignificantly lower ratio of aberration types than random
scattering of tracks. More critical assessment of experimental
data also does not support this theory [L54, S66]; inparticular, reexamination of extensive plant data has ruled out
both this theory and any LET-dependence in the ratio ofinterstitial deletions to inter-arm exchanges [S72]. A different
high-LET fingerprint has more recently been proposed by
Lucas [L55], who suggested that the proportion of unrejoined(“incomplete”) chromosome aberrations will be greater withhigh-LET radiation, because the local densityof DNA doublestrand breaks is greater than for low-LET radiation andcompetition between broken ends leads to a greater chance ofincomplete exchange events. Published data are cited that givea ratio of incomplete exchanges to complete exchanges(translocations) of about 9 for low-LET radiations and about
2 for high-LET radiations, with "mixed-LET" radiations
giving intermediate values. While this theory has yet toreceive critical appraisal in the literature, it may suffer fromthe same problems as the earlier theory, insofar as onlycertain
sets of data give large differences between high- and low-LET
radiations, partly because of difficulties in correctly defining
incomplete aberrations. There may be further difficulties with
the idea that competition between broken ends will necessarily
lead to more incomplete aberrations and with the simple
assumption that incomplete aberrations represent unrejoined
breaks.

E. NOVEL MECHANISMS OF
GENETIC CHANGE
195. Most of the processing of DNA damage by cellular
repair enzymes is completed within a few hours of irradiation, including the fixation of mutations. However, there isevidence that cellular responses continue to occur for much
longer periods, over many cell generations. These responses
include delayed cell death and genetic changes. While these
responses may in part be attributed to the time taken for thecell to recover from irradiation, there are in principle several
reasons why persistent effects could occur. These include
persistence of the damaging agent; persistence of certain
forms of DNA damage, i.e. lack of repair; the repair ofdamage leading to rearrangements of the genome, whichthemselves upset the correct functioning of the cell (e.g.
“position effects” on blocks of genes); and the induction of a41

long-lived metabolic disturbance in somatic cells, such thatenzymatic activities (e.g. DNA polymerases) involved in the
fidelity of maintaining the genome do not function properly.
196. It has been known for many years that cells may takesome time to die following irradiation [E2, J7], but more
recently emphasis has been placed on lethal mutations that
may take effect many cell generations after irradiation [B53,
G9, S16]. These two phenomena may in part be aspects of thesame response. Delay in cell death in fibroblastic cell lines
may be explained by the time taken for cells to show theeffects of loss of essential genes through chromosome
fragmentation (see paragraph 110). As cells divide, theysegregate broken chromosomes (often seen as micronuclei),and the encoded gene products are diluted out of daughter
cells, eventually causing death [J7]. In the same way, lethal
mutations may cause late-onset death in cells; however, they
can arise through more subtle effects on the genome thansimple chromosomal fragmentation and loss. Thus, all typesof mutation, from point mutation to large genetic changes,
may represent lethal mutations if they lead to loss of essential
gene products. However, to account for the high frequency oflethal mutations at long times after irradiation, it has also
been proposed that some form of persistent genetic instability
can be induced by radiation.
197. In addition to delayed death, the mutation frequency
in inessential genes such as HPRT has been found to bepersistently elevated in a large fraction of clones surviving
irradiation [C20]. In these experiments, cells irradiatedwith x rays (12 Gy) were grown as separate clones andexamined for both the proportion of cells surviving and formutant frequency at different times after irradiation. It was
found that clones showing reduced survival also commonly
had elevated mutant frequencies at the HPRT gene for as
many as 50-100 cell generations following irradiation. The
mutant frequency in individual clones was highly variable
but sometimes exceeded 1 10-3. These delayed mutations
appeared to be predominantly point mutations [L34].
198. Chromosomal aberrations have also been found to persist following irradiation. One-cell mouse embryos irradiatedwith x rays or neutrons showed an approximately linear
increase in the frequency of chromosomal aberrations per cellin the first, second, and third mitoses post-irradiation. The
relatively high frequency of aberrations, especially forneutrons, and the occurrence of chromatid-type aberrations on
the third mitosis following irradiation suggested that new
aberrations were being produced in post-irradiation cell cycles[W40, W41]. Similar results were obtained with x irradiationof two-cell mouse embryos [W42]. Delayed chromosomal
aberrations were not observed in embryos treated with restriction endonucleases, suggesting that lesions other than DNA
double-strand breaks are responsible for this effect [W48]. A
significant increase in chromosome- and chromatid-type
aberrations was also found in cell cultures derived from foetalskin biopsies of mice x irradiated as zygotes [P25]. The studyof chromosomal aberrations in lymphocyte and fibroblastclones surviving x irradiation has similarly given evidence of
persistent genetic effects [H30, M34, P33]. Analysis of clones

42

ANNEX F: DNA REPAIR AND MUTAGENESIS

for two months following irradiation showed that >20% had
sporadic aberrations as well as transmissable clonal karyotype
alterations [H31].
199. Genetic instability in clones of cells surviving
irradiation has also been described for alpha-particle irradiation of cultured haematopoietic stem cells from CBA/H mice[K8]. In this case, chromosomal aberrations were measured incell clones surviving 3 Gy from x rays or 0.25-1 Gy fromalpha particles (0.5 Gy from 238Pu alpha particles corresponds
to an average of one track per cell). About 50% of the clones
surviving alpha-particle irradiation carried aberrations; thesewere mostly non-identical chromatid-type aberrations,suggesting that they had arisen many generations after
irradiation. The frequency of such delayed aberrationinduction in x-ray survivors was only about 2% [K8]. This
form of chromosomal instability in bone marrow cells was
also found to be transmissible in vivo, by transplanting malecells irradiated with alpha particles into female recipients[W38]. The repopulated haemopoietic system showed
instability persisting for up to one year. Alpha particles havealso been shown to induce similar delayed chromosomal
effects in the bone marrow of two out of four normal humans[K9]. It was suggested that the lack of effect in someindividuals reflects genetic determinants that vary in thehuman population, and additional studies of other inbred
mouse strains have also been found to show varying levels of
this form of genetic instability. The alpha-particle-induced
instability was, however, found to be independent of the p53
status of the cell [K32].
200. Heavy ion (neon, argon, or lead) irradiations have also
been found to induce chromosomal instability in cultured
human fibroblasts [M35, S53]. Analysis of mass cell cultures
for up to 25 passages following irradiation showed that thefrequency of aberrations declined at first but then increased
until >60% of the cells showed aberrations. In contrast to
clonal cell populations, these aberrations showed that thetelomeric regions of specific chromosomes (1, 13, and 16)
were involved. In a study of alpha particles of different LET,
the frequency of micronuclei was found to be increased overthat in unirradiated hamster cells seven days after irradiation,even at a dose giving one alpha-particle traversal per cell
nucleus [M36]. Calculations indicate that the target size forthis effect exceeds the size of the nucleus, suggesting that
direct DNA damage by radiation tracks is not causal.
201. It has been speculated that these events indicate thationizing radiation may induce an “untargeted” mechanism ofmutagenesis in cells, as a result of the epigenetic alteration of
enzymatic pathways controlling genomic stability [L18]. The
idea that DNA may not require a direct hit from radiation tohave an increased frequency of genetic changes has received
support from the measurement of non-mutational responses.
With reference to the process of carcinogenesis in particular,
the work of Kennedy et al. [K10, K11] suggests that radiation
may induce high-frequency events that predispose the cell
towards further (spontaneously-occurring) changes in theprocess of malignant cell transformation. Furthermore, a
study of the induction of sister-chromatid exchanges inimmortalized hamster cells by very low doses of 238Pu alphaparticles claimed that induction could be measured at doses(0.3 mGy) where <1% of the cell s nuclei were traversed by analpha-particle track [N8]. Subsequent studies of primary
human fibroblasts confirmed this finding; low doses of alphaparticles give three times as many sister-chromatid exchanges[D13] or a five times higher HPRT gene mutation frequency[N25] than predicted from the number of nuclei traversed by
the particle tracks, giving a considerably larger target size forthis effect than expected from nuclear dimensions [D13].
Similarly, the use of a physical barrier to protect one part of acell population from alpha-particle irradiation showed the
expected reduction in killing of mouse bone-marrow stem
cells but did not reduce chromosomal instability [L63]. These
data are considered as evidence for the existence of a
“bystander” effect; that is, damage signals may be transmitted
from irradiated to neighbouring unirradiated cells. In arefinement of this type of experiment, an alpha-particle
microbeam was used to give precise irradiation of thecytoplasm of cells, without damaging their nuclei. Under
these conditions, an average increase of three-fold was foundin the mutation frequency in a sensitive human-hamster
hybrid cell line (paragraph 171), and the mutation spectrum
was similar to that occurring spontaneously [W51]. One
explanation of this phenomenon is that reactive oxygen
species are generated by radiation in the whole cell andperhaps also in the surrounding medium, and these species or
stable reaction products (e.g. lipid peroxides) diffuse into the
nucleus and persist to cause chromosomal effects. Evidence
for this idea has been found with x or neutron irradiation ofbone marrow cells; various indicators of persistent oxygen
radical activity were found in cell cultures seven days after
irradiation [C40]. It has also been shown that alpha particles
can produce factors in culture medium or in cells that cause
increases in sister-chromatid exchanges, as noted above; thesefactors are inhibited by superoxide dismutase, an enzyme thatcatalyses the conversion of superoxide ions produced in waterradiolysis [L17]. Similarly, in the microbeam experiments,
reactive oxygen species were implicated in the mutagenic
effects of cytoplamsic irradiation [W51]. Further, in humanfibroblasts, the modulation of proteins involved in the p53dependent pathway (Section II.B.2) following very low dosesof alpha particles occurs in more cells than have been
traversed by an alpha-particle track, apparently through cellcell contact [A24]. The potential consequences of bystander
effects are discussed further in Annex G, “Biological effects
at low radiation doses”.
7

202. In an attempt to link the delayed appearance of chromosomal aberrations to cancer-proneness, Ponnaiya et al. [P24]measured this form of instability in strains of mice differingin their sensitivity to radiation-induced mammary cancer.
Strikingly, cells from the more sensitive strain (BALB/c)showed a marked increase in the frequency of chromatidaberrations after 16 population doublings, while the lesssensitive strain (C57BL/6) showed no increase in aberrations
over the control level.
203. Higher-than-average frequencies of specific types ofchromosomal aberrations have been found after irradiation of

ANNEX F: DNA REPAIR AND MUTAGENESIS

strains of mice that are prone to certain types of cancer. These
aberrations are not necessarily the delayed effects of irradiation but nonetheless may indicate that radiation damage to
specific parts of the genome contributes disproportionately tocancer induction. As an example, interstitial deletions ofmouse chromosome 2 are consistently associated withradiation-induced acute myeloid leukaemia in several inbred
strains of mice. The chromosome breakpoints involved inthese chromosome 2 deletions are non-randomly distributedand can be detected at high frequency at early times following
irradiation, suggesting that they may be an early event inleukaemogenesis [B54, B55, H43, H45, R28, T23]. Investigation of two mouse strains showing large differences insusceptibilityto radiation-induced thymic lymphomas showedthat at early times following x irradiation the incidence ofspecific chromosomal aberrations, especially trisomy 15, wasmuch higher in the cancer-prone strain than in the resistantstrain [C21]. These examples suggest that certain chromosomal regions may be more sensitive to radiation-induced
genetic changes that are associated with cancer, although it isdifficult to quantifythis contribution precisely without a better
understanding of the development of aberrant clones of cells
in animal tissues.
204. In some mutation systems very high frequencies ofmutation occur both spontaneously and after irradiation. With
radiation induction, the amount of initial damage (e.g. thenumbers of DNA double-strand breaks per unit dose) in themutational target may not in these cases be sufficient toaccount for the numbers of mutations induced. While it ispossible to consider that every type of damage (paragraph 15,
Table 1) may sometimes lead to mutation, these examples ofhigh-frequency mutation have usually been proposed to arise
from a form of genetic instability. For instance, somatic
mutation frequencies of coat colour in Pink-eyed unstablemice are linear with x-ray dose down to 10 mGy and occur atleast 100 times more frequently per unit dose than germ-line
mutations in the mouse specific-locus tests [S57]. The Pinkeyed unstable male mice have a reduction in the pigment of
the coat and eyes, caused by gene duplication interfering withnormal pigment production; deletion of this duplication isscored as the mutation, giving normal pigmentation seen as
black spots on the grey coat.
205. The spontaneous frequency of mutation at tandemrepeat (VNTR or “minisatellite”) DNA sequences in the germline of mice is also very high (1%-10% in offspring). The
mechanism of mutation at these hypervariable loci does notappear to involve unequal exchange between homologous
chromosomes [W30] but rather some form of complex geneconversion process [J10]. The incorporation of humantandem-repeat sequences into yeast cells, to study themechanisms of variability, has confirmed that such sequences
are destabilized in meiosis and that this process depends on
the initiation of homologous recombination at a nearby DNA
double-strand break [D23]. Irradiation of mouse spermatogonial stem cells by x rays increased the mutation frequency
in offspring relative to controls [D10]. More extensive
experiments at different stages of spermatogenesis using both
single- and multi-locus probes showed no increase in mutation

43

frequency in post-meiotic spermatids but gave a doubling doseof 0.33 Gy for premeiotic spermatogonial and stem cells. The
dose-response for mutation induction by 0.5-1 Gy x rays,
combined for spermatogonial and stem cells, was linear[D19]. In contrast, in a separate study using one of the sameprobes at different germ-cell stages, the irradiation of
spermatogonia gave a non-significant increase in mutation
frequency, while the frequency in irradiated spermatids was
significant. The increase in mutation frequency was not linearwith dose, showing little increase above 1 Gy [F5, S19].
Similar data were reported for 252Cf irradiations (35% gamma
rays, 65% neutrons): spermatids again showed the highest
induced mutation frequency, and a single dose (1 Gy) tospermatogonia was significantly mutagenic. The RBE forthese mutations with 252Cf was 5.9 for spermatogonia, 2.6 forspermatids, and 6.5 for spermatogonial stem cells [N15].
More work is required to reconcile the discrepancies that are
seen for different germ-cell stages. Recently a striking result
has been found from tandem-repeat mutation studies following irradiation of male mice with 0.5 Gy 252Cf neutrons, andthe mating of these mice to unirradiated mice through twogenerations to yield second-generation (F2) progeny.
Remarkably, the F2 mice showed an elevated frequency ofmutation in the repeat sequences inherited from both male andfemale lines (6-fold and 3.5-fold, respectively), suggesting thatgenetic instability can be transmitted through the germline[D24]. The induced mutation frequencyat these hypervariablesequences seems to be too high, by two orders of magnitude,
for direct damage by radiation at the sites of mutation; it was
proposed that some indirect mechanism of mutation induction
is responsible [D19, F5, S19, D24].
206. Human spontaneous germ-line mutation frequencies attandem-repeat loci can also be high (1%-7% per gamete). Ina pilot study of children of survivors of the atomic bombings(mean gonad dose, generally to only one of the parents =1.9 Sv) and matched controls, mutation frequency wasmeasured at six tandem-repeat loci. The average mutation
frequency was similar in the two groups, at 1.5% per gamete
per locus for the exposed gametes and 2% for the unexposed
gametes [K26]. A further study of the same children using aprobe to detect multiple-repeat loci again showed no increasein mutation rate for the exposed group [S67]. However, atwofold increase in the frequency of mutation at tandemrepeat loci has been reported for children of parents resident
in the Mogilev district of Belarus, which was heavily
contaminated in the Chernobyl accident [D14]. Four tandemrepeat loci were tested, three of which showed increases by afactor of 1.7-2.0, while one locus (in which only one mutation
was found) showed a reduction in the exposed group. The 79
families tested were compared to a control Caucasian
population from the United Kingdom, which showed asimilar overall distribution of repeat lengths at these loci. Ina follow-up study using five additional tandem-repeat loci andincluding a further 48 families from Mogilev, the samegeneral twofold increase was found when compared with thesame (United Kingdom) control population [D1]. The meandose to the exposed population was calculated to be
27.6±3.3 mSv from 137Cs; some evidence for a dose-response
relationship was obtained by dividing the exposed group into

44

ANNEX F: DNA REPAIR AND MUTAGENESIS

those receiving >20 mSv (mutation rate = 0.024) and thosereceiving <20 mSv (rate = 0.018), compared with the control
rate of 0.011. While it is clear that there is a general increasein the mutation rate for several different genomic sites in the
exposed population, difficulties of interpretation exist becauseof the geographical disparity of the control group and thepossibility that other environmental agents may be
responsible for the increased mutation rate [S67]. As a direct
test of the involvement of tandem-repeat loci in radiationinduced carcinogenesis, normal and tumour DNA from postChernobyl thyroid carcinomas was examined for mutations inthree loci [N22]. Mutations were found in 3 of 17 tumours(18%), with one of these having mutations in all three loci,while none of 20 sporadic thyroid cancers from patients
without a historyof radiation exposure showed tandem-repeat
mutations.
207. Recent studies of “hypermutation” in non-dividing
cultures of bacteria placed under stress have also suggested
that recombination processes are involved in the formation ofsmall deletions at high frequency [R29]. It is suggested thatwhen bacteria are starved of nutrients, for example, mutations
arise at a very high rate in order to survive (if sufficientmutations occur, one of these may be sufficiently favourable
to allow the cells to adapt to the prevailing conditions).
Evidence supports a model in which cells enter a transient
hypermutable state in which DNA double-strand breaks
initiate homologous recombination activity (see Figure Id),
priming error-prone DNA synthesis. It seems likely that theerrors (mutations) arise as a result of a down-regulation of themismatch repair system (paragraph 166) [H44]. Similarly,
specific types of oxidative base damage (e.g. 8-oxoguanine,
paragraph 24) may lead to small deletion mutations whenmismatch correction is compromised [B56]. The significanceof these findings is that the intrinsic mutability of cells is
modifiable, and this principle may apply equally to many
types of cell, perhaps including those involved in carcinogenesis [R30].
208. Other reported phenomena mayalso be connected to the
induction of genetic instability following irradiation. One ofthese is coincident mutation: in cells selected for radiationinduced mutation at one genomic site, a high frequency ofmutations occurs at other sites. Li et al. [L16] found two
mutations at tandem-repeat loci among 50 x-ray-induced TK
gene mutants, a frequency of 4 10-2 and far in excess of
expectation based on current knowledge of radiation-induced
gene mutation frequencies. No second-site mutations were
found in 70 unirradiated clones. Perhaps the clearest example
of coincident mutations was found in an extensive study of
x-ray-induced mutation in the fungus Neurospora crassa[D5]. In a chromosomal region containing 21 genes, about
10% of the radiation-induced mutants had mutations in morethan one gene following a single acute dose. However, a yeast
cell study has revealed a possible mechanistic basis for nontargeted mutations that occur relatively close to the initial siteof damage, without invoking genetic instability[S52]. A DNA
double-strand break was placed at a single site in one yeast
chromosome, using a site-specific endonuclease, andmutations were measured in a gene situated adjacent to thebreak site. After the break had undergone recombination
repair, it was found that the adjacent gene had sustained a
300-fold increase in the frequency of point mutations. This
result suggests that the break repair process is error-prone,
presumably because of a lack of fidelity in DNA repair
synthesis, and extends over distances of several hundred DNA
base pairs.
209. It is difficult to know how the reports of delayed genetic
effects and instability following both low- and high-LET
radiation may apply to humans. As indicated above (paragraphs 201-203), a high frequency of radiation-induced
genetic or epigenetic changes may contribute to cancer
incidence, at least in those instances where an accumulationof somatically-stable changes is required. However, it is still
not clear whether genetic instability or a higher-than-normal
mutation rate is a necessary for the development of tumours[T38]. In the case of inherited genetic alterations, where germ
cells are the target, measurements ofmutation frequencymade
at short intervals after irradiation could underestimate theinduced mutation frequency. However, in the case of alphaparticle irradiation, mice injected with 239Pu and subsequently
mated to tester stocks (specific-locus method) over manyweeks were not found to show increasingly high levels ofmutation [R16]. It is, of course, possible that some potential
increase in mutations may be balanced by selection against
sperm carrying an increased load of genetic damage due toinstability. The type of mutation induced may be important; if
these are point mutations, they could have a greater chance oftransmission and could lead to dominant genetic effects inoffspring. The potential importance of genetic instability incarcinogenesis, especially at low radiation doses, is discussed
further in Annex G, “Biological effects at low radiation
doses”.

F. MUTATION FREQUENCIES ANDCONSEQUENCES
210. Radiation-induced mutations are always measured
against a background of spontaneously-occurring mutations. The mechanisms of spontaneous mutation are
numerous; the chemical reactivity of DNA leads toinstability, and there are inherent errors in replicating avery large molecule. Some of these mechanisms will
overlap those of radiation damage; for example, oxidative
damage from metabolic processes in aerobic organisms will
give both base damage and strand breaks (paragraph 19).
This indicates why many of the types of mutation that
occur spontaneously are similar to those formed by ionizing radiation. The cell requires efficient repair processes tocope with endogenous damage; if unrepaired, the damage
will lead to base-pair substitutions as well as some largerchanges. The increase in mutant frequency found after
exposure to ionizing radiation is likely to come from boththe additional load of damage similar to that occurring
spontaneously (such as DNA base damage and loss) andmore complex radiation-induced damage that cannot be
handled easily by the cell's battery of repair enzymes(Section I.B).

ANNEX F: DNA REPAIR AND MUTAGENESIS

211. As was stressed in Section IV.B, an important
influence on the observed frequency of mutation is thetolerance of the genome site to large changes. When largechanges are tolerated, the spectrum of mutations inducedby ionizing radiation shows that at least half (andcommonly more than half) of the mutations measured
shortly after irradiation are large deletions and rearrangements. This spectrum may change with time after
irradiation, but as yet there are too few data to comment
meaningfullyon this possibility. Mutation frequencies havebeen measured for a few target genes, which have beenchosen for their ease of use and especially for theirpresence in a hemizygous state. While these measurements

45

seem unlikely to represent the genome as a whole (seeSection IV.E), they do give some idea of the variation in
frequencies and the reasons for this variation. An
interesting comparison can be made of the hamster APRTand HPRT genes; since both of these code for inessential
enzymes of similar function (purine salvage) they shouldboth detect all types of mutant. However, the APRT gene isthought to have an essential gene situated downstream,
which limits the possibility of detecting very large
deletions (paragraph 179). Table 4 shows that thefrequencies of both spontaneous and radiation-induced
APRT mutations are lower than those for HPRT by morethan an order of magnitude.

Table 4
Mutant frequency and spectrum in two different genes of hamster cells

Agent

Mutant frequency(106 cells)

Number analysed

Deletions/rearrangements(%)

APRT (autosomal hemizygous) [M13]
None
EMS a
Gamma rays

0.13
430
1.53.0 b

125
48
85

7.2
0
22.3

HPRT (X-linked hemizygous) [T20]
None
EMS a
Gamma rays c
a
b
c

6.2
690
38.8

44
56
48

18
0
71

Ethylmethane sulphonate, an alkylating agent.
Doses 2.5 and 4 Gy.
Dose 5 Gy (both EMS and gamma-ray doses gave 20% cell survival).

212. Whether available mutation systems are sensitive
enough to yield useful data on genetic damage in situationsof practical importance is questionable. There has beensome controversy over attempts to measure, for example,
the frequency of HPRT gene mutations in the blood
lymphocytes of radiotherapy technicians and patients. It
was concluded that some of these studies are better atrevealing the variables involved in measuring mutation at
low doses than at giving reliable data on mutation
frequencies (reviewed in [T22]). One study [N16], however, included molecular analysis of the mutations found inlymphocytes of radioimmune therapy patients and showedthat a higher proportion with large genetic changes
occurred than in controls, with some dose dependence forthe fraction of mutants with large changes due to cumulative 131I activity. Studies of the survivors of the atomic
bombings [H32, H33] reported a slight increase, about 10%
per Gy, in HPRT mutant frequency as estimated dose
increased. This frequency increase is considerably lower
than found in freshly-irradiated lymphocytes, suggestingthat HPRT mutants are selected against over the long time
period involved. However, it has recently been found thata significant increase in HPRT mutant frequency could be
detected in combined data from 142 liquidators involved in
the Chernobyl accident (24% increase in the liquidator
group relative to Russian controls) after adjustment for age
and smoking [T49].

213. The problem of selection against mutant cells seems tobe less severe in some more recently developed mutation
assays. An assay based on the loss of one copy of the cellsurface marker glycophorin A, encoded by the GPA gene, inhuman erythrocytes gave dose-dependent increases in mutant
frequency in survivors of the atomic bombings, and thefrequencies per unit dose were similar to those found in
human cells irradiated with low-LET radiation underlaboratory conditions [K1, K33, L44, L45]. In contrast to the
HPRT data, there was a positive correlation between thefrequencies of chromosomal aberrations and GPA mutants in
exposed individuals; additionally, survivors with malignant
solid tumours showed a significantly higher mutant frequency
than those without cancer. This assay will measurechromosome loss (by, for example, non-disjunction) as well as
mutation and recombination events in individuals alreadyheterozygous for the GPA gene (about half of the humanpopulation), but it has the drawback that no molecularanalysis of the mutations is possible (mature erythrocytes lack
nuclei) [G24]. The GPA mutation system has also shown
dose-dependent mutation induction in individuals exposed todoses of up to 6 Gy during or following the Chernobyl
accident, and the slope of the dose response was very similar
for these measurements and the GPA mutants measured insurvivors of the atomic bombings (at about 25 10-6 mutants
Gy-1) [J18]. This result has received support from subsequent
measurements of GPA mutation in liquidators [W53] orANNEX F: DNA REPAIR AND MUTAGENESIS

214. Where enough measurements have been made, theaverage frequency of gene mutation induced by low-LET
radiation is similar in cells from different somatic tissues
and species. This can be illustrated by a plot of induced
HPRT mutant frequency against surviving fraction (Figure
VIIIa), where differences in intrinsic radiosensitivity aretaken into account [T13, T14]. The plot also indicates that
there is some consistency in the relationship between
mutation and killing, suggesting that these responses
derive from similar types of damage, a constant fraction ofwhich is converted to mutations [T17]. It should be notedthat this relationship does not say anything about theabsolute mutant frequencies for different tissues ororganisms; these may differ substantially, as seen, forexample, in the response of different germ-cell stages in
the mouse [S29].
215. The mutation frequency/survival plot can also be used
to show that both the type of radiation and cellularparameters can influence the effectiveness of mutationinduction. Thus, for HPRT mutation, densely ionizing
radiation shows an increase in the effectiveness by a factor of
no more than 2; i.e. the RBE for mutation is about twice thatfor cell killing (Figure VIIIb) [C22, T16, T18]. Similarly,
x rays vary in effectiveness with the phase of the cell cycle; anincrease in the effectiveness of mutation induction by factorsof 2-3 is found relative to cell killing, especially in G1/S phase[B14, J6]. It is tempting to speculate that the repair systems
operational at this point in the cycle (see Section II.B.1) are
more prone to recombinational errors.
216. Mutation induction is also subject to dose-rate effects; ingeneral, the effectiveness of low-LET radiations is reduced byfactors of 2-4 at low dose rates [T22]. However, it has also
been found in specific conditions that the mutagenic
effectiveness may remain the same or may increase at low
dose rates relative to high dose rates [T22]. For example, if
TK6 lymphoblastoid cells are exposed to low dose rates, nochange in mutagenic effectiveness is found [K36], while theirmore radiation-resistant counterpart WIL2-NS (paragraph185) shows an approximately twofold reduction in effectiveness [F11]. Lack of alteration in mutagenic effectiveness withdose rate has also been seen for radiation-sensitive (DNArepair-deficient) cell lines [F12]. In addition, at dose rates of
low-LET radiation of 0.5 mGy min-1, rodent cell lines may

show no dose-rate effect [E10, E11, F13] or an increased(inverse) dose-rate effect [C44, C45]. Also, inverse dose-rate
effects have been seen with low dose rates of high-LET
radiation [K37, N18].
20

Human
Hamster
Mouse

18
16
MUTANTS PER 105 CELLS

people living in the vicinity of Chernobyl [L71] at the time of
the accident. However, use of the GPA system to measuremutation in more than 700 Estonian and Latvian workers
involved in the Chernobyl clean-up, with estimated mediandoses of about 100 mGy, did not detect a consistent increasein mutant frequency [B39]. The mutagenic effects of 5.3 MeV
alpha particles (average LET = 140 keV µm-1) in a human
exposed to 232Th over a 43-year period following thorotrast
injection were measurable using the GPA gene assay [L11].
These data showed a fivefold increase in GPA mutants andcorrelated to large increases in chromosomal aberration
frequency in lymphocytes from the same individual. Other
data associated with the consequences of the Chernobyl
accident are considered in the Annex J, “Exposures andeffects of the Chernobyl accident”.

14
12
10
8
6
4
2

Figure VIIIa00.1
SURVIVING FRACTION

1

14

0.01

Alpha particles
x rays12
MUTANTS PER 105 CELLS

46

( )

10
8
6
4
2

Figure VIIIb
0
1

0.5

0.2
SURVIVING FRACTION

0.1

0.05

Figure VIII. Mutation-survival relationship for HPRT
genes.
VIIIa: Similarity of induced mutant frequency in celllines from human, mouse and hamster [T17];
VIIIb: The increased effectiveness of densely ionizing
radiation (238Pu alpha particles) compared to x rays inhamster cells [T18].

217. Some of these dose-rate data point to specific cellular
processes influencing the response; in particular, it appears
that the response is dependent on the capacity of cells to
repair damage to DNA [T22]. It has long been thought that
cellular repair capacity is responsible for the reduction in
mutagenic effectiveness with dose rate, as seen for example inclassical mouse germ-cell data [R6, R7]. Where little or nodose-rate effect is found, the repair capacity of the genetic
region or of the whole cell is therefore likely to be impaired.
This situation may also apply in some germ-cell stages;
spermatozoa of both Drosophila and the mouse show no doserate effect [R6, T31]. The finding that very low dose rates givelittle relative effect, or an inverse effect, in some somatic celllines may indicate that the full repair capacity of a cellrequires a minimum level of insult (damage induced per unit

ANNEX F: DNA REPAIR AND MUTAGENESIStime) before it is brought to bear on that damage. However, it
should be noted that chronic irradiation of mouse
spermatogonial stem cells has shown no further reduction in
specific-locus mutation frequency below 8 mGy min-1 withdose rates down to 7 µGy min-1 [R17].

G. SUMMARY
218. DNA damage caused by ionizing radiations leads tovarious types of mutation: the smaller mutations, such as basepair substitutions, appear to result from damage to single
bases, while larger changes, such as large deletions andrearrangements, probably arise from DNA double-strand
breakage. The spectrum of radiation-induced mutations isdominated by the larger molecular changes if the genetic site
assessed will tolerate these without lethality. In view of thepropensityto induce large genetic changes, it maybe supposedthat the average radiation-induced mutation has greater
consequences in terms of extensive alterations of the genome
than the average spontaneous mutation or those induced byother agents. As yet no distinct mutational fingerprint has
been identified for ionizing radiations, although there is asuggestion that the more complex types of molecular change
may be overrepresented in the spectrum. High-LET radiations
induce a higher mutation frequency per unit dose, but there
are insufficient data to establish whether the mutation
spectrum is different from that for low-LET radiations.
219. Attempts by cellular enzymes to repair DNA damage
are intimately linked to mutation formation. The molecularmechanisms of radiation-induced mutation include illegitimate recombination, but where genes are heterozygous thereis evidencethat homologous recombination is involved (giving

47

loss of heterozygosity, commonly seen in some cancers as loss
oftumour-suppressor gene function). Some forms ofradiationinduced base damage will be repaired by mismatch repair
pathways; loss of mismatch repair function can lead to
hypermutability, which is linked to specific forms of cancer
such as hereditary non-polyposis colon cancer. The involvement of repair processes in mutation formation suggests thatthe intrinsic mutability of cells is not fixed but will vary with
their repair capacity; this concept may have importantconsequences for the process of carcinogenesis.
220. There are several reports of persistent genetic effects incells and animals following irradiation, suggesting thatgenetic instability is induced. A possible explanation for thisphenomenon is that stable oxidative reaction products persist
and can continue to give genetic damage in subsequent cell
generations, although other explanations are possible (paragraph 195). In some mutation systems very high frequencies
of mutation (hypermutability) occur spontaneously and after
irradiation; on the basis of target size, it is unlikely that thesemutations are induced directlybyradiation damage at the sitesof mutation. Hypermutability also suggests that some form ofgenetic instability is induced by radiation treatment, but atpresent it is difficult to know how these data relate toresponses such as cancer induction.
221. Methods have been established for measuring mutations
with some accuracy in human somatic cells, such as blood
lymphocytes, but most mutation systems lack the sensitivity to
be used as indicators of genetic damage in cases of low-doseradiation exposure. Some success has been found in measuring mutant frequencies in cells of individuals exposed torelatively high doses of radiation, such as atomic bomb
survivors and Chernobyl recovery operation workers.

CONCLUSIONS
222. DNA repair processes have evolved in all biologicalorganisms to combat the deleterious effects of damage to their
genetic material. Attempts to repair DNA damage also cause
many types of mutation, through inability to properly restore
the DNA sequence. Many of the genes involved in the repair
of DNA damage in human cells have now been cloned, andtheir functional analysis has led to considerable progress in
our understanding of the ways in which cells and organisms
respond to radiation damage. Several different pathways of
repair are required to cope with damage from ionizing
radiation (Figure III), and the consequences of losing repair
capacity can be drastic. The importance of the repair genefunction has been revealed in particular by the development ofmethods to knock out specific genes in experimental animals.
Using these methods, it has been found that the loss of repair
gene activity is often lethal in early stages of development(Table 2). Where the knockout animals survive the loss of
repair capacity, they are commonly very prone to cancer.
These findings reinforce more limited studies with rareindividuals in the human population, which have suggested
for some time that radiosensitivity is linked to cancer

proneness. The converse finding, that mice lacking
homologues ofthe human breast-cancer-predisposing (BRCA)
genes are radiosensitive, is a further important illustration ofthis link. Loss of repair gene function is considered to lead tocancer proneness primarily through an increase in genetic
instability, although the mechanistic details of this process
remain to be elucidated. The precise contribution of loss of
repair gene function to the risk of radiation-induced cancer inthe population is unknown at present; if calculations are
restricted to those rare individuals with recognized repair
syndromes, then this contribution will be small, but it isalready known that a more subtle variation in these genes
occurs widely in the human population.
223. Cellular survival assays indicate a twofold variation in
the response of individuals in the general population to acuteradiation exposure. This variation may become three- or
fourfold when irradiation is given at low dose rates. Specificgroups of individuals may show a consistently elevatedsensitivity; for example, individuals heterozygous for theataxia-telangiectasia-mutated (ATM) gene, who constitute

48

ANNEX F: DNA REPAIR AND MUTAGENESIS

about 1 percent of the population, show enhanced radiosensitivity especially when tested in a chromosome-damage
assay. This finding has recently been reinforced by similar
data with transgenic mice heterozygous for ATM deficiency.
Strikingly, using the chromosome-damage assay, one study
has shown that about 40 percent of breast cancer patientsshow a similar enhancement of radiation sensitivity. Recent
evidence shows that the enhanced radiosensitivity of breast
cancer patients has a genetic basis; it has been suggested that
the ATM gene is involved in predisposition to breast cancer,
but the evidence for this is controversial. It is possible that
small reductions in the efficiency of any one of a number ofgenes involved in radiation response, due to subtle mutations
or polymorphisms, will account for the existence of radiosensitive groups of individuals.
224. The analysis of DNA repair processes has revealed thatthey are part of a complex response system in our cells, which
includes genes involved in recognizing and signalling thepresence of damage and genes operating checkpoints toensurethat cells do not progress through the cell cycle if they carry
DNA damage. The functional analysis of repair genes
involved in radiation response has also revealed a link
between radiosensitivity and immune dysfunction, becausesome of the gene products involved in the repair of radiationinduced DNA double-strand breaks also assemble functional
immune genes. Thus, radiosensitivity in humans can arise
from the loss of a broader spectrum of gene functions than
was initially recognized.
225. A reduction in DNA repair capacity may have several
consequences for the cellular radiation response, including
alteration in the shapes of dose-response curves, the loss of
low-dose-rate sparing, and a loss of relative effectiveness forhigh-LET radiations. It is probable that these consequences
arise mainlyfrom an alteration in the abilityto repair complex
forms of DNA damage, particularly those involving doublestrand breaks, since this type of damage is extremely
hazardous to the cell. DNA repair capacity is therefore an
important component of radiation dose-response, and it isclear that it needs to be considered as a variable in modellingradiation action, especially when attempting to extrapolate to
low dose exposure. However, before the effects of repair
pathways can be modelled accurately, there is still considerably more to learn about the way in which different pathways
contribute to the overall response to radiation. At present it is
not known how cells control the use of different repair pathways in responding to damage, and in particular how abalance is achieved between correct (error-free) repair andrepair leading to mutation induction. Additionally, more
information is needed on the relationship between DNA
repair and apoptosis in different cells and tissues to predict theoutcome of radiation exposure.
226. There is experimental evidence that pre-irradiation withlow doses (5-10 mGy) can increase the resistance of cells to a
subsequent higher dose, and that a more sensitive cellularresponse to radiation exists at low doses than at higher doses.
These observations have led to some controversy; they suggestthat under some circumstances a resistance factor can be
induced by low radiation doses, but the mechanistic basis of

the observations has proved difficult to establish. It has also
been found in separate studies that a number of different genes
and proteins are induced or repressed by radiation, although
few of these appear to be involved directly in the repair of
DNA damage. To date little of this characterization has been
carried out in a systematic fashion, because it has been
conditioned by the availability of cloned genes and proteins.
However, new technology based on large-scale genesequencing coupled to micro-arraying of sequences isbeginning to revolutionize this type of study by enabling theassay of hundreds or thousands of gene products at one time.
By examining the levels of many gene products from cells
before and after irradiation, as well as with time elapsed
following irradiation, it will be possible to see how thedamage response is coordinated. These methods also permit
a molecular description of the differences in levels of geneproducts present in normal and diseased tissues, including
tumour tissue, facilitating an understanding ofthe mechanistic
basis of cancer induction byagents such as ionizing radiation.
227. Many different types of DNA damage are caused byionizing radiations, ranging from isolated single-strand
damage at sites of single ionizations to complex DNA alterations at sites of clustered ionizations. The more complex
forms of damage may be unique to the interaction of ionizing
radiations with DNA, compared with damage occurring spontaneously or that caused by other DNA-damaging agents.
Several attempts have been made to establish whether radiation damage can lead to a distinct fingerprint of genetic changes, but this has proved elusive. It is clear, for example, thatthe spectrum of gene mutation arising from radiation damage
has differences from the spontaneous mutation spectrum, but
the overlap in the two spectra is considerable. Again, newtechnologies based on fluorescent in situ hybridization, the useof reporter genes with fluorescent tags, and comparative
genomic hybridization will allow a refined view of radiationinduced genetic changes and the hope of future distinctions.
228. Many genetic changes caused by radiation occur withina few hours of giving the dose, but there is experimental
evidence to show that some forms of change may occur after
much longer times and following many cell divisions. In
addition, at some sites in the human genome, the frequency ofradiation-induced genetic changes is much higher than would
be expected based on direct damage to DNA by radiation
tracks. These observations broaden our knowledge of themechanisms of radiation action, but elucidation is required
further before the consequences of these mechanisms forradiation risk can be understood.
229. It is clear that much more knowledge of the structure of
the human genome, in particular the disposition of the genes
within it and their responses to radiation, is required before it
will be possible to predict the average frequency of mutationinduced by a given dose of radiation. Additionally, the rangeof repair capacities present in the human population,
including carriers of defective repair genes, will have to beconsidered to predict mutability on an individual basis. A
more complete knowledge of these response mechanisms willallow greater accuracy in the prediction of radiation-induced
carcinogenic and hereditary effects.

ANNEX F: DNA REPAIR AND MUTAGENESIS

49

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7

ANNEX F: DNA REPAIR AND MUTAGENESIS

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L18

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