Methods for mapping genetic mutations

The present invention relates to a method for detecting base sequence differences within homologous regions of two DNA molecules comprising the steps of contacting at least one strand of the first DNA molecule with the complementary strand of the second DNA molecule under conditions such that base pairing occurs, contacting the resulting DNA duplexes with a protein that recognizes substantially all base pair mismatches under conditions such that the protein forms specific complexes with its cognate mispairs, and detecting the resulting DNA:protein complexes by a suitable analytical method. Also disclosed are protein components of DNA mismatch correction systems and the use of these components in methods for genetic mapping.

FIELD OF THE INVENTION 
The present invention relates to methods for mapping genetic differences 
among deoxyribonucleic acid ("DNA") molecules, especially mutations 
involving a difference in a single base between the base sequences of two 
homologous DNA molecules. More specifically, this invention relates to 
such mapping methods which employ proteins that recognize and correct 
mismatched DNA base pairs in double-stranded DNA. This invention also 
relates to the manufacture and use of certain novel products enabled by 
the identification and isolation of proteins that are components of 
mismatched base pair recognition and correction systems. 
BACKGROUND OF THE INVENTION 
Mapping of genetic differences between individuals is of growing importance 
for both forensic and medical applications. For example, DNA 
"fingerprinting" methods are being applied for identification of 
perpetrators of crimes where even small amounts of blood or sperm are 
available for analysis. Biological parents can also be identified by 
comparing DNAs of a child and a suspected parent using such means. 
Further, a number of inherited pathological conditions may be diagnosed 
before onset of symptoms, even in utero, using methods for structural 
analyses of DNA. Finally, it is notable that a major international effort 
to physically map and, ultimately, to determine the sequence of bases in 
the DNA encoding the entire human genome is now underway and gaining 
momentum in both institutional and commercial settings. 
DNA molecules are linear polymers of subunits called nucleotides. Each 
nucleotide comprises a common cyclic sugar molecule, which in DNA is 
linked by phosphate groups on opposite sides to the sugars of adjoining 
nucleotides, and one of several cyclic substituents called bases. The four 
bases commonly found in DNAs from natural sources are adenine, guanine, 
cytosine and thymine, hereinafter referred to as A, G, C and T, 
respectively. The linear sequence of these bases in the DNA of an 
individual encodes the genetic information that determines the heritable 
characteristics of that individual. 
In double-stranded DNA, such as occurs in the chromosomes of all cellular 
organisms, the two DNA strands are entwined in a precise helical 
configuration with the bases projecting inward and so aligned as to allow 
interactions between bases from opposing strands. The two strands are held 
together in precise alignment mainly by hydrogen bonds which are permitted 
between bases by a complementarity of structures of specific pairs of 
bases. This structural complementarity is determined by the chemical 
natures and locations of substituents on each of the bases. Thus, in 
double-stranded DNA, normally each A on one strand pairs with a T from the 
opposing strand, and, likewise, each G with an opposing C. 
When a cell undergoes reproduction, its DNA molecules are replicated and 
precise copies are passed on to its descendants. The linear base sequence 
of a DNA molecule is maintained in the progeny during replication in the 
first instance by the complementary base pairings which allow each strand 
of the DNA duplex to serve as a template to align free nucleotides with 
its polymerized nucleotides. The complementary nucleotides so aligned are 
biochemically polymerized into a new DNA strand with a base sequence that 
is entirely complementary to that of the template strand. 
Occasionally, an incorrect base pairing does occur during replication, 
which, after further replication of the new strand, results in a 
double-stranded DNA offspring with a sequence containing a heritable 
single base difference from that of the parent DNA molecule. Such 
heritable changes are called genetic mutations, or more particularly in 
the present case, "single base pair" or "point" mutations. The 
consequences of a point mutation may range from negligible to lethal, 
depending on the location and effect of the sequence change in relation to 
the genetic information encoded by the DNA. 
The bases A and G are of a class of compounds called purines, while T and C 
are pyrimidines. Whereas the normal base pairings in DNA (A with T, G with 
C) involve one purine and one pyrimidine, the most common single base 
mutations involve substitution of one purine or pyrimidine for the other 
(e.g., A for G or C for T), a type of mutation referred to as a 
"transition". Mutations in which a purine is substituted for a pyrimidine, 
or vice versa, are less frequently occurring and are called 
"transversions". Still less common are point mutations comprising the 
addition or loss of a single base arising in one strand of a DNA duplex at 
some stage of the replication process. Such mutations are called single 
base "insertions" or "deletions", respectively, and are also known as 
"frameshift" mutations, due to their effects on translation of the genetic 
code into proteins. Larger mutations affecting multiple base pairs also do 
occur and can be important in medical genetics, but their occurrences are 
relatively rare compared to point mutations. 
Mapping of genetic mutations involves both the detection of sequence 
differences between DNA molecules comprising substantially identical 
(i.e., homologous) base sequences, and also the physical localization of 
those differences within some subset of the sequences in the molecules 
being compared. In principle, it is possible to both detect and localize 
limited genetic differences, including point mutations within genetic 
sequences of two individuals, by directly comparing the sequences of the 
bases in their DNA molecules. In practice, however, direct DNA sequencing 
has highly restricted usefulness for mapping mutations due to the major 
time and effort required to determine the sequence of even one DNA 
fragment comprising a few hundred base pairs. Typically, a single 
functional unit of genetic information, a gene, may be encoded in tens of 
thousands of base pairs of human chromosomal DNA. Thus comparing the 
sequence of a complete gene from one individual with that of another by 
direct DNA sequencing involves analyses of multiple short fragments of 
that gene, requiring many months if not years of effort. It may also be 
noted that there are estimated to be hundreds of thousands of genes in the 
entire human gene complement or genome, as it is called, any one of which 
may be involved in some genetically determined disease. 
Accordingly, several simpler methods for detecting differences between DNA 
sequences have been developed which although providing less direct 
information about base sequence differences, nevertheless do yield useful 
observations under limited circumstances. For example, some pairs of 
single-stranded DNA fragments with sequences differing in a single base 
may be distinguished by their different migration rates in electric 
fields, as in denaturing gradient gel electrophoresis. This method does 
not detect all the possible single-base differences between DNA fragments 
and is restricted to fragments comprising at most a few hundred base 
pairs. Further, it is technically difficult to generate consistent 
analyses using this method. Thus this approach has extremely limited 
utility for detection and localization of single base sequence differences 
between DNAs encoding whole genes. 
DNA restriction systems found in bacteria, for example, comprise proteins 
which generally recognize specific sequences in double-stranded DNA 
composed of 4 to 6 or more base pairs. In the absence of certain 
modifications (e.g., a covalently attached methyl group) at definite 
positions within the restriction recognition sequence, endonuclease 
components of the restriction system will cleave both strands of a DNA 
molecule at specific sites within or near the recognition sequence. Such 
short recognition sequences occur by chance in all natural DNA sequences, 
once in every few hundred or thousand base pairs, depending on the 
recognition sequence length. Thus, digestion of a DNA molecule with 
various restriction endonucleases, followed by analyses of the sizes of 
the resulting fragments (e.g., by gel electrophoresis), may be used to 
generate a physical map ("fingerprint") of the locations in a DNA molecule 
of selected short sequences. 
It is well known in the art that comparisons of such restriction maps of 
two homologous DNA sequences can reveal differences within those specific 
sequences that are recognized by those restriction enzymes used in the 
available maps. Restriction map comparisons may localize any detectable 
differences within limits defined ultimately by the resolving power of DNA 
fragment size determination, essentially within about the length of the 
restriction recognition sequence under certain conditions of gel 
electrophoresis. To achieve such resolution in location of a point 
mutation by restriction mapping, however, all fragments resulting from 
digestion with each restriction nuclease must be within a range of 
distinguishable sizes, usually below an upper limit of between 10 and 20 
thousand base pairs (kbp), and preferably less than one kbp, using 
standard gel electrophoresis techniques. Since each different restriction 
enzyme scans only a fraction of a percent of all the sequences in any DNA 
molecule, literally thousands of analyses with thousands of different 
enzymes would be needed to completely compare two DNAs encoding even one 
gene, assuming that enzymes recognizing all possible 4 to 6 base sequences 
were known, which they are not. 
In practice, selected heritable differences in restriction fragment lengths 
(i.e., restriction fragment length polymorphisms, "RFLP"s) have been 
extremely useful, for instance, for generating physical maps of the human 
genome on which genetic defects may be located with a relatively low 
precision of hundreds or, sometimes, tens of thousands of base pairs. 
Typically, RFLPs are detected in human DNA isolated from small tissue or 
blood samples by using radioactively labeled DNA fragments complementary 
to the genes of interest. These "probes" are allowed to form DNA duplexes 
with restriction fragments of the human DNA after separation by 
electrophoresis, and the resulting radioactive duplex fragments are 
visualized by exposure to photographic (e.g., X-ray sensitive) film, 
thereby allowing selective detection of only the relevant gene sequences 
amid the myriad of others in the genomic DNA. 
When the search for DNA sequence differences can be confined to specific 
regions of known sequence, the recently developed "polymerase chain 
reaction" ("PCR") technology can be used to reduce the amount of effort 
needed to detect and locate a single base difference as compared to the 
usual DNA sequencing approach which requires molecular cloning of the DNA 
fragment of interest. Briefly, this method utilizes short DNA fragments 
complementary to sequences on either side of the location to be analyzed 
to serve as points of initiation for DNA synthesis (i.e., "primers") by 
purified DNA polymerase. The resulting cyclic process of DNA synthesis 
results in massive biochemical amplification of the sequences selected for 
analysis, which then may be easily detected and, if desired, further 
analyzed, for example, by restriction mapping or direct DNA sequencing 
methods. In this way, selected regions of a human gene comprising a few 
kbp may be amplified and examined for sequence variations, but only in 
cases where sequences spanning a particular location of interest are 
known. 
In clinical practice, the PCR method is of limited utility, for example, in 
detection of known heritable variants of selected human genes which differ 
by only one or a few specific base pairs (i.e., allelic forms a gene). For 
example, the human .beta.-globin gene comprises several alleles that can 
be distinguished by this approach; but the overall utility is highly 
limited, particularly when faced with a need to detect sequence 
differences which may be scattered over large stretches of a gene, as in 
the diagnosis of conditions resulting from frequent new mutational events 
in human populations, in the Lesch-Nyan syndrome, for example. 
Another known method for detecting and localizing single base differences 
within homologous DNA molecules involves the use of a radiolabeled RNA 
fragment with base sequence complementary to one of the DNAs and a 
nuclease that recognizes and cleaves single-stranded RNA. The structure of 
RNA is highly similar to DNA, except for a different sugar and the 
presence of uracil (U) in place of T; hence, RNA and DNA strands with 
complementary sequences can form helical duplexes ("DNA:RNA hybrids") 
similar to double-stranded DNA, with base pairing between A's and U's 
instead of A's and T's. It is known that the enzyme ribonuclease A ("RNase 
A") can recognize some single pairs of mismatched bases (ie., "base 
mispairs") in DNA:RNA hybrids and can cleave the RNA strand at the mispair 
site. Analysis of the sizes of the products resulting from RNase A 
digestion allows localization of single base mismatches, potentially to 
the precise sequence position, within lengths of homologous sequences 
determined by the limits of resolution of the RNA sizing analysis (Myers, 
R. M. et al., 1985, Science, 230, 1242-1246). RNA sizing is performed in 
this method by standard gel electrophoresis procedures used in DNA 
sequencing, an approach which limits the practical resolution to mapping 
of single base mispairs in a DNA:RNA hybrid comprising an RNA of only 
several hundred nucleotides. Moreover, this RNAse A method requires 
preparing complementary RNA probes from each DNA sequence to be examined, 
which requires more work and is more technically demanding than methods 
using only DNA (such as restriction mapping). Further, RNase A does not 
efficiently recognize all possible mispairings of DNA and RNA bases, 
resulting in a significant inefficiency in detection of all point 
differences between DNA sequences. 
It has also been reported that S1 nuclease, an endonuclease specific for 
single-stranded nucleic acids, can recognize and cleave limited regions of 
mismatched base pairs in DNA:DNA or DNA:RNA duplexes. Therefore, it has 
been suggested that S1 nuclease could be used to map single base pair 
differences between DNA molecules by sizing of cleavage fragments. 
However, more extensive analysis of this enzyme has established that a 
mismatch of at least about 4 consecutive base pairs actually is generally 
required for recognition and cleavage of a duplex by S1 nuclease, thus 
precluding its use for detection of any point mutations. 
Thus, none of the available methods for comparing the base sequences of 
DNAs, other than direct sequencing, can efficiently detect and localize 
all possible single base differences. Further, all of these methods, 
including especially DNA sequencing, require substantial labor and 
repetitive analyses with various sequence specific reagents (e.g., 
multiple nucleases or short nucleic acid strands) to detect all single 
base differences within two specimens of a single human gene. 
Hence, there is a need for simpler and more efficient approaches, both for 
detecting and for localizing genetic differences between DNA sequences to 
facilitate both clinical diagnoses and forensic investigations. In 
particular, the observations above indicate a specific need for simpler 
and more efficient methods and reagents for detection of any possible 
single base differences between long DNA sequences, for example, between a 
complete gene from one individual and the entire genome of another. There 
is also a further need for simpler methods for localization of any 
possible single base differences within the sequences of homologous 
regions of long DNA molecules such as those encoding one or more complete 
genes and comprising several kbp of DNA. 
The present invention contemplates the use of certain proteins that 
recognize mismatched base pairs in double-stranded DNA (and, therefore, 
are called "mispair recognition proteins") in defined systems for 
detecting and mapping point mutations in DNAs. Accordingly, it is an 
object of the present invention to provide methods for using such mispair 
recognition proteins, alone or in combination with other proteins, for 
detecting and localizing single base differences between DNA molecules, 
particularly those DNAs comprising several kbp. Additionally, it is an 
object of this invention to develop modified forms of mispair recognition 
proteins to further simplify methods for identifying specific bases which 
differ between DNAs. 
Enzymatic systems capable of recognition and correction of base pairing 
errors within the DNA helix have been demonstrated in bacteria, fungi and 
mammalian cells, but the mechanisms and functions of mismatch correction 
are best understood in Escherichia coli. Of the several mismatch repair 
systems that have been identified in E. coli, the most relevant here is 
the methyl-directed pathway for repair of DNA biosynthetic errors. The 
fidelity of DNA replication in E. coli is enhanced 100-1000 fold by this 
postreplication mismatch correction system. This system processes base 
pairing errors within the helix in a strand-specific manner by exploiting 
patterns of DNA methylation. Since DNA methylation is a postsynthetic 
modification, newly synthesized strands temporarily exist in an 
unmethylated state, with the transient absence of adenine methylation on 
GATC sequences directing mismatch correction to new DNA strands within the 
hemimethylated duplexes. 
In vivo analyses in E. coli have shown that selected examples of each of 
the different mismatches are subject to correction with different 
efficiencies. G-T, A-C, G-G and A-A mismatches are typically subject to 
efficient repair. A-G, C-T, T-T and C-C are weaker substrates, but well 
repaired exceptions exist within this class. It is thought that the 
sequence environment of a mismatched base pair may be an important factor 
in determining the efficiency of repair in vivo. The mismatch correction 
system is also capable in vivo of correcting differences between duplexed 
strands involving a single base insertion or deletion. Further, genetic 
analyses have demonstrated that the mismatch correction process requires 
intact genes for several proteins, including the products of the mutH, 
mutL and mutS genes, as well as DNA helicase II and single-stranded DNA 
binding protein (SSB). 
The present inventors have been seeking to identify and isolate specific 
proteins that are required for correction of mismatched base pairs and to 
understand the specific biochemical functions of these mispair correction 
system components. The products of the mutH and mutS genes have been 
purified to near homogeneity in biologically active form. Analysis of the 
MutH protein has suggested that it functions in strand discrimination by 
incising the unmethylated DNA strand at GATC sites. The isolated MutS 
protein has been shown to recognize four of the eight possible mismatched 
base pairs (specifically, G-T, A-C, A-G and C-T mispairs; Su, S. -S. and 
Modrich, P., 1986, Proc. Nat. Acad. Sci. U.S.A., 84, 5057-5061). The 
hierarchy of apparent affinities of isolated MutS protein for the 
particular examples of the four mispairs tested in these studies did not 
correlate well with in vivo efficiencies of mismatch correction. Hence, 
these studies left undetermined whether or not additional proteins, acting 
alone or in concert with MutS, are required for or influence the 
recognition of other base mispairs. 
SUMMARY OF THE INVENTION 
It has now been discovered that a single DNA base mispair recognition 
protein can form specific complexes with any of the eight possible 
mismatched base pairs embedded in an otherwise homologous DNA duplex. It 
has also been revealed that another mispair recognition protein can 
recognize only one specific base pair mismatch, A-G, and in so doing, it 
chemically modifies a nucleotide at the site of the mispair. In addition, 
defined in vitro systems have been established for carrying out 
methyl-directed mismatch repair processes. Accordingly, the present 
invention contemplates the use of such mispair recognition proteins and 
related correction system components to detect and to localize point 
mutations in DNAs. 
For clarity in the following discussion, it will be useful to point out 
here certain distinctions related to the fact that some proteins that 
recognize DNA base mispairs are merely DNA binding proteins, while others 
modify the DNA as a consequence of mispair recognition. Notwithstanding 
the fact that in the latter situation the protein modifying the DNA may be 
associated with the DNA only transiently, hereinafter, whether a mispair 
recognition protein is capable of DNA binding only or also of modifying 
DNA, whenever it is said that a protein recognizes a DNA mispair, this is 
equivalent to saying that it "forms specific complexes with" or "binds 
specifically to" that DNA mispair in double-stranded DNA. In the absence 
of express reference to modification of DNA, reference to DNA mispair 
recognition does not imply consequent modification of the DNA. Further, 
the phrase "directs modification of DNA" includes both cases wherein a DNA 
mispair recognition protein has an inherent DNA modification function 
(e.g., a glycosylase) and cases wherein the mispair recognition protein 
merely forms specific complexes with mispairs, which complexes are then 
recognized by other proteins that modify the DNA in the vicinity of the 
complex. Finally, it should be noted in the following discussion that 
those DNA base mispairs (e.g., A-G or C-C) which are recognized by a given 
protein are referred to as the "cognate" base mispairs for that protein. 
Accordingly, the present invention relates to a method for detecting base 
sequence differences within homologous regions of two DNA molecules 
comprising the steps of contacting at least one strand of the first DNA 
molecule with the complementary strand of the second DNA molecule under 
conditions such that base pairing occurs, contacting the resulting DNA 
duplexes with a protein that recognizes substantially all base pair 
mismatches under conditions such that the protein forms specific complexes 
with its cognate mispairs, and detecting the resulting DNA:protein 
complexes by a suitable analytical method. 
In the practice of a preferred embodiment of this aspect of this invention, 
the mispair recognition protein is the product of the mutS gene of E. coli 
or another functionally homologous protein, and an advantageous analytical 
method for detecting the DNA:protein complex comprises the steps of 
contacting the DNA:protein complexes with a selectively adsorbent agent, 
such as a membranous nitrocellulose filter, under conditions such that 
protein:DNA complexes are retained on the agent while DNA not complexed 
with protein is not retained, and measuring the amount of DNA in the 
retained complexes. Other suitable analytical methods for detecting the 
DNA:protein complex are disclosed. 
In addition to methods designed merely to detect base sequence differences 
between DNAs, this invention further relates to a method for both 
detecting and localizing individual base sequence differences within 
homologous regions of two DNA molecules comprising the steps of contacting 
at least one strand of the first DNA molecule with the complementary 
strand of the second DNA molecule under conditions such that base pairing 
occurs, contacting the resulting double-stranded DNA duplexes with a 
protein that recognizes at least one base mispair under conditions such 
that the protein forms specific complexes with its cognate mispairs and 
thereby directs modification of at least one strand of the DNA in the 
resulting DNA:protein complexes in the vicinity of the DNA:protein 
complex, and determination of the location of the resulting DNA 
modification relative to a known sequence within the homologous regions of 
the DNAs by a suitable analytical method. 
In the practice of one embodiment of this aspect of this invention, the 
mispair recognition protein is the product of the mutS gene of E. coli or 
is another functionally homologous protein; the step in which the DNA is 
modified in the vicinity of the DNA:protein complex further comprises 
contacting the DNA:MutS protein complex with a defined E. coli DNA 
mismatch correction system under conditions such that single-stranded gaps 
are produced in the vicinity of the complexed protein; and the method for 
determining the locations of these single-stranded gaps within the DNA 
duplex comprises the steps of cleaving the DNA with a single-strand 
specific endonuclease and at least one restriction endonuclease, and 
comparing the electrophoretic mobilities of the resulting modified DNA 
fragments with DNA restriction fragments not contacted with the defined 
mismatch correction system. Suitable single-strand specific endonucleases 
include the S1 single-strand specific nuclease, for example, or other 
functionally similar nucleases well known in the art. 
The present invention further relates, in part, to forms of mispair 
recognition proteins which have been altered to provide an inherent means 
for modifying at least one strand of the DNA duplex in the vicinity of the 
bound mispair recognition protein. 
In a principal embodiment of this aspect of this invention, the altered 
mispair recognition protein is the modified product of the mutS gene of E. 
coli or is another functionally homologous modified protein to which is 
attached an hydroxyl radical cleaving function; and the DNA modification 
step in the DNA mispair localization method further comprises contacting 
this modified protein with the DNA in under conditions such that the 
radical cleaving function cleaves at least one strand of the DNA in the 
vicinity of the protein. Additional altered forms of mispair recognition 
proteins that modify at least one strand of the DNA in a DNA:protein 
complex in the vicinity of the bound protein are disclosed. 
The present invention also comprises another E. coli DNA mispair 
recognition protein that recognizes only A-G mispairs without any apparent 
requirement for hemimethylation. This protein, the product of the mutY 
gene, is a glycosylase which specifically removes the adenine from an A-G 
mispair in a DNA duplex. Accordingly, this MutY protein is useful for the 
specific detection of A-G mispairs according to the practice of the 
present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS 
The present invention relates to a method for detecting base sequence 
differences within homologous regions of two DNA molecules comprising the 
steps of contacting at least one strand of the first DNA molecule with the 
complementary strand of the second DNA molecule under conditions such that 
base pairing occurs, contacting the resulting DNA duplexes with a protein 
that recognizes at least one base pair mismatch under conditions such that 
the protein forms specific complexes with its cognate mispairs, and 
detecting the resulting DNA:protein complexes by a suitable analytical 
method. 
In the practice of this method, the two DNA molecules to be compared may 
comprise natural or synthetic sequences encoding up to the entire genome 
of an organism, including man, which can be prepared by well known 
procedures. Detection of bases sequence differences according to this 
method of this invention does not require cleavage (by a restriction 
nuclease, for example) of either of the two DNAs, although it is well 
known in the art that rate of base pair formation between complementary 
single-stranded DNA fragments is inversely related to their size. This 
detection method requires that base sequence differences to be detected 
lie within a region of homology constituting at least about 14 consecutive 
base pairs of homology between the two DNA molecules, which is about the 
minimum number of base pairs generally required to form a stable DNA 
duplex. Either one or both of the strands of the first DNA may be selected 
for examination, while at least one strand of the second DNA complementary 
to a selected first DNA strand must be used. The DNA strands, particularly 
those of the second DNA, advantageously may be radioactively labeled to 
facilitate direct detection, according to procedures well known in the 
art. 
Methods and conditions for contacting the DNA strands of the two DNAs under 
conditions such that base pairing occurs are also widely known in the art. 
In the practice of a principal embodiment of this aspect of this invention, 
the mispair recognition protein is the product of the mutS gene of E. 
coli. Preparation of this protein substantially free of other proteins has 
been reported previously (Su, S. -S. and Modrich, P., 1986, Proc. Nat. 
Acad. Sci. U.S.A., 84, 5057-5061, which is hereby incorporated herein by 
reference). 
The surprising ability of the MutS protein to recognize examples of all 
eight single base pair mismatches within double-stranded DNA, even 
including C-C mispairs which do not appear to be corrected in vivo, is 
demonstrated by the fact that MutS protein protects DNA regions containing 
each mismatch from hydrolysis by DNase I (i.e., by "DNase I footprint" 
analyses), as recently reported (Su, S. -S., et al., 1988, J. Biol. Chem., 
263, 6829-6835). The affinity of MutS protein for the different mispairs 
that have been tested varies considerably. Local sequence environment may 
also affect the affinity of the MutS protein for any given base mispair; 
in other words, for example, the affinity for two specific cases of A-C 
mispairs, which are surrounded by different sequences, may not be the 
same. Nevertheless, no examples of base mispairs have been found that are 
not recognized by isolated MutS protein. Accordingly, it is believed that 
this method of this invention detects substantially all possible single 
base differences between homologous regions of any two DNA molecules. 
It should be particularly noted that the DNA duplexes which MutS recognizes 
are not required to contain GATC sequences and, hence, they do not require 
hemimethylation of A's in GATC sequences, the specific signal for the full 
process of mispair correction in vivo; therefore, use of MutS in this 
method allows recognition of a DNA base mispair in DNAs lacking such 
methylation, for instance, DNAs isolated from human tissues. 
A protein which appears to be functionally and in part, at least, 
structurally homologous to the E. coli MutS protein has also been 
discovered in a methyl-directed mispair correction system in Salmonella 
typhimurium bacteria (Pang et al., 1985, J. Bacteriol., 163, 1007-1015). 
The gene for this protein has been shown to complement E. coli strains 
with mutations inactivating the mutS gene and the amino acid sequence of 
its product shows homology with that of the E. coli MutS protein. 
Accordingly, this S. typhimurium protein is also believed to be suitable 
for the practice of this aspect of the present invention. Other organisms, 
including man, are known to possess various systems for recognition and 
repair of DNA mispairs, which, as one skilled in the art would appreciate, 
comprise mispair recognition proteins functionally homologous to the MutS 
protein. Accordingly, it is believed that such DNA base mispair 
recognition proteins are also suitable for use in the present invention. 
In the practice of a preferred embodiment of this aspect of this invention, 
an advantageous analytical method for detecting the DNA:protein complex 
comprises the steps of contacting the DNA:protein complexes with a 
selectively adsorbent agent, such as a membranous nitrocellulose filter, 
under conditions such that protein:DNA complexes are retained on the agent 
while DNA not complexed with protein is not retained, and measuring the 
amount of DNA in the retained complexes. Absent radioactive labeling of at 
least one strand used to form the DNA duplexes, the DNA in complexes on 
the filter may be detected by any of the usual means in the art for 
detection of DNA on a solid substrate, including annealing with 
complementary strands of radioactive DNA. 
The nitrocellulose filter method for detecting complexes of MutS protein 
with base mispairs in DNA has been reported in detail (Jiricny, J. et al., 
1988, Nuc. Acids Res. 16, 7843-7853, which is hereby incorporated herein 
by reference). Besides simplicity, a major advantage of this method for 
detecting the DNA:protein complex over other suitable methods is the 
practical lack of a limitation on the size of DNA molecules that can be 
detected in DNA:protein duplexes. Therefore, this embodiment of this 
method is useful for detecting single base sequence differences between 
DNA fragments as large as can be practically handled without shearing, at 
least 50 kbp. 
Another suitable analytical method for detecting the DNA:protein complex 
between the mispair recognition protein and a cognate mispair in a DNA 
duplex comprises the steps of separating the DNA:protein complexes from 
DNA that does not form such complexes on the basis of electrophoretic 
mobility, and detecting the DNA in the less mobile DNA:protein complexes. 
The DNA in the DNA:protein complexes may be detected by any of the usual 
standard means for detection of DNA in gel electrophoresis, including 
staining with dyes or annealing with complementary strands of radioactive 
DNA. Detecting complexes comprising the MutS base mispair recognition 
protein and mispairs in DNA duplexes is also described in the foregoing 
reference (Jiricny, J. et al., 1988, Nuc. Acids Res., 16, 7843-7853). 
Under the usual conditions employed in the art for detecting specific 
DNA:protein complexes by gel electrophoresis, complex formation of a 
protein with a double-stranded DNA fragment of up to several hundred base 
pairs is known to produce distinguishable mobility differences. 
Other suitable analytical methods for detecting the DNA:MutS protein 
complex include immunodetection methods using an antibody specific for the 
base mispair recognition protein. For example, antibodies specific for the 
E. coli MutS protein have been prepared readily by standard immunological 
techniques. Accordingly, one immunodetection method for complexes of MutS 
protein with DNA comprises the steps of separating the DNA:protein 
complexes from DNA that does not form such complexes by 
immunoprecipitation with an antibody specific for MutS protein, and 
detecting the DNA in the precipitate. According to the practice of this 
aspect of this invention, quantitative immunoassay methods known in the 
art may be employed to determine the number of single base mispairs in 
homologous regions of two DNA molecules, based upon calibration curves 
that can be established using complexes of a given mispair recognition 
protein with DNA duplexes having known numbers of mispairs. 
In addition to methods that merely detect base sequence differences, this 
invention further relates to a method for both detecting and localizing 
individual base sequence differences within homologous regions of two DNA 
molecules comprising the steps of contacting at least one strand of the 
first DNA molecule with the complementary strand of the second DNA 
molecule under conditions such that base pairing occurs, contacting the 
resulting double-stranded DNA duplexes with a protein that recognizes at 
least one base mispair under conditions such that the protein forms 
specific complexes with its cognate mispairs and thereby directs 
modification of at least one strand of the DNA in the resulting 
DNA:protein complexes in the vicinity of the DNA:protein complex, and 
determination of the location of the resulting DNA modification relative 
to a known sequence within the homologous regions of the DNAs by a 
suitable analytical method. 
In the method of the present invention for localization of single base 
differences, there is provided a suitable means for modifying at least one 
strand of the DNA duplex in the vicinity of the bound mispair recognition 
protein. The modification may be any alteration for which there is a means 
of detection, for instance a chemical modification including breaking of a 
chemical bond resulting in, as examples, cleavage between nucleotides of 
at least one DNA strand or removal of a base from the sugar residue of a 
nucleotide. Specific means for modifying DNAs in the vicinity of the 
DNA:protein complex are provided below for several embodiments of this 
aspect of the invention, together with interpretations of the phrase "in 
the vicinity of", as appropriate to the practical limitations of the 
modification approach in each instance. 
In the practice of one embodiment of this aspect of this invention, the 
mispair recognition protein is the product of the mutS gene of E. coli or 
is another functionally homologous protein; and the step in which the DNA 
is modified in the vicinity of the DNA:protein complex further comprises 
contacting the DNA:MutS protein complex with a defined E. coli DNA 
mismatch correction system under conditions such that single-stranded gaps 
are produced in the vicinity of the complexed protein. 
The complete defined mismatch correction system comprises the following 
purified components: E. coli MutH, MutL, and MutS proteins, DNA helicase 
II, single-strand DNA binding protein, DNA polymerase III holoenzyme, 
exonuclease I, DNA ligase, ATP, and the four deoxynucleoside 
triphosphates. This set of proteins can process seven of the eight 
base-base mismatches in a strand-specific reaction that is directed by the 
state of methylation of a single GATC sequence located 1 kilobase from the 
mispair. This defined system is described further in Example 1, below. It 
should be noted that the lack of ability to repair C-C base mispairs in 
this embodiment of this aspect of the present invention is not a major 
limitation of the method for detecting all possible base sequence 
differences between any two naturally occurring DNA sequences because 
mutations apparently due to C-C mispairing during DNA replication appear 
arise most infrequently in vivo. 
For the purposes of generating single-stranded gaps in the vicinity of the 
DNA:MutS protein complexes, DNA duplexes containing mispaired base pairs 
are contacted with the defined mismatch correction system under the 
standard conditions described in Example 1, Table 3 (Complete reaction), 
except for the following differences: exogenous dNTP's are omitted or, 
preferably, 2',3'-dideoxynucleoside-5'-triphosphates (ddNTPs) are added at 
100 uM with dNTPs at 10 uM, to inhibit repair of single-strand gaps; and 
DNA ligase may be omitted from the reaction. The requirement for 
methyl-directed strand incision by MutH may be obviated by provision of a 
single-stand nick by some other means within the vicinity of the mispair, 
as described in Example 1, FIG. 5. A suitable means for inducing such 
nicks in unmethylated DNA is limited contact with a nuclease, DNase I, for 
example; under conditions that are well known in the art, this approach 
creates nicks randomly throughout double-stranded DNA molecules at 
suitable intervals for allowing the mispair correction system to create 
single-stranded gaps in the vicinity of a mispair anywhere in the DNA. 
It should be noted that in this embodiment of this method for localizing 
mismatch base pairs, "in the vicinity of" a base mispair is defined 
practically by the size of the single-strand gaps typically observed under 
the above conditions, namely up to about one kbp from the mismatch base 
pair. Further, in this embodiment, the method for determining the 
locations of these single-stranded gaps within the DNA duplex comprises 
the steps of cleaving the DNA with a single-strand specific endonuclease 
and at least one restriction endonuclease, and comparing the 
electrophoretic mobilities of the resulting modified DNA fragments with 
DNA restriction fragments not contacted with the defined mismatch 
correction system. Suitable single-strand specific endonucleases include 
the S1 single-strand specific nuclease, for example, or other functionally 
similar nucleases well known in the art. Additional restriction mapping 
may be performed as needed to further localize any fragment modifications 
observed in initial applications of the method, until, if desired, a 
restriction fragment of convenient size for direct sequence determination 
is obtained for direct comparisons of sequences of the two DNA molecules 
in the vicinity of the base sequence difference. 
The present invention further relates, in part, to forms of mispair 
recognition proteins which have been altered to provide an inherent means 
for modifying at least one strand of the DNA duplex in the vicinity of the 
bound mispair recognition protein. 
In a principal embodiment of this aspect of this invention, the altered 
mispair recognition protein is the modified product of the mutS gene of E. 
coli or is another functionally homologous modified protein to which is 
attached an hydroxyl radical cleaving function; and the DNA modification 
step in the DNA mispair localization method further comprises contacting 
this modified protein with the DNA under conditions such that the radical 
cleaving function cleaves at least one strand of the DNA in the vicinity 
of the protein. 
Several methods for attaching an hydroxyl radical cleaving function to a 
DNA binding protein are known in the art. For example, lysyl residues may 
be modified by chemically attaching the 1,10-phenanthroline-copper complex 
to lysine residues, resulting in conversion of a DNA binding protein into 
a highly efficient site-specific nuclease that cleaved both DNA strands 
(in the presence of hydrogen peroxide as a coreactant) within the 20 base 
pair binding site of the protein, as determined by DNase I footprinting 
(C. -H. Chen and D. S. Sigman, 1987, Science, 237, 1197). Chemical 
attachment of an EDTA-iron complex to the amino terminus of another DNA 
binding protein similarly produced a sequence specific DNA cleaving 
protein that cut both strands of the target DNA within a few bases of 
recognition site of similar size (J. P. Sluka, et al., 1987, Science, 235, 
777). 
An alternate means for attaching the hyroxyl radical cleaving function to 
this same protein involved extension of the amino terminus with the three 
amino acids, Gly-Gly-His, which is consensus sequence for the 
copper-binding domain of serum albumin (D. P. Mack et al., 1988, J. Am. 
Chem. Soc., 110, 7572-7574). This approach allows for preparation of such 
an artificial DNA cleaving protein directly by recombinant methods, or by 
direct synthesis using standard solid phase methods, when the peptide is 
sufficiently short as it was in this case (55 residues including the 3 
added amino acids), thereby avoiding the need for an additional chemical 
modification step of the reagent which is both time consuming and 
difficult in large scale production. In contrast to the EDTA-iron complex, 
the particular peptide sequence constructed in this instance cleaved only 
one example out of four recognition sites in different sequence 
environments. 
Nevertheless, one skilled in the art of protein engineering would 
appreciate that this general approach for converting a DNA binding protein 
into a DNA cleaving protein by attachment of an hydrogen radical cleavage 
function is widely applicable. Hence, DNA base mispair recognition 
proteins which normally only bind to DNA are modified to cleave DNA by 
attachment of an hydroxyl radical cleavage function, according to the 
practice of this aspect of this invention, without undue experimentation, 
by adjustment of appropriate variables taught in the art, particularly the 
chemical nature and length of the "spacer" between the protein and the 
metal binding site. 
In the DNA sequence localization method according to this embodiment which 
employs a modified DNA base mispair recognition protein with attached 
hydroxyl radical cleavage function, the means for modification of the 
DNA:protein complex is a suitable metal ion and associated cofactor or 
cofactors, and the modification comprises double-stranded cleavage of the 
DNA within the vicinity of any cognate base mispair wherein the "vicinity" 
substantially corresponds to the sequence of DNA protected by the binding 
of the protein to a base mispair, generally within about 20 base pairs. A 
single-strand specific nuclease, S1, for instance, may be used to augment 
cleavage by the modified base mispair recognition protein in the event 
that a single-strand bias is suspected in the cleavage of any DNAs with 
which the protein forms a specific complex. 
Additional altered forms of mispair recognition proteins that modify at 
least one strand of the DNA in a DNA:protein complex in the vicinity of 
the bound protein according to the present invention include proteins 
comprising the portions or "domains" of the unmodified base mispair 
recognition enzymes that are essential for binding to a DNA mispair. These 
essential domains comprise peptides in the unmodified protein which are 
made resistant to proteolytic digestion by formation of specific 
DNA:protein complexes at cognate DNA base mispairs. These essential DNA 
binding domains further comprise peptide sequences that are most highly 
conserved during evolution; such conserved domains are evident, for 
example, in comparisons of the sequences of the E. coli MutS protein with 
functionally homologous proteins in S. typhimurium and other structurally 
similar proteins. Accordingly, peptide sequences of a DNA base mispair 
recognition protein that are protected from proteases by formation of 
specific complexes with mispairs in DNA and, in addition or in the 
alternative, are evolutionarily conserved, form the basis for a 
particularly preferred embodiment of this aspect of the present invention, 
since such peptides constitute less than half the mass of the intact 
protein and, therefore, are advantageous for production and, if necessary, 
for chemical modification to attach a cleavage function for conversion of 
the DNA binding protein into a DNA cleavage protein specific for sites of 
DNA base mispairs. 
The present invention also comprises another E. coli DNA mispair 
recognition protein that recognizes only A-G mispairs without any 
requirement for hemimethylation. This protein, the product of the mutY 
gene, is a glycosylase which specifically removes the adenine from an A-G 
mispair in a DNA duplex. The Mut Y protein has been purified to near 
homogeneity by virtue of its ability to restore A-G to C-G mismatch 
correction to cell-free extracts (K. G. Au et al., Proc. Nat. Acad. Sci. 
U.S.A., 85, 9163, 1988) of a mutS mutY double mutant strain of E. coli, as 
described in Example 2, below. It is a 36 kDa polypeptide that apparently 
exists as a monomer in solution. MutY, an AP endonuclease, DNA polymerase 
I, and DNA ligase are sufficient to reconstitute MutY-dependent, A-G to 
C-G repair in vitro. A DNA strand that has been depurinated thusly by the 
MutY protein is susceptible to cleavage by any of several types of AP 
(apurinic) endonuclease (e.g., human AP endonuclease II) or by piperidine, 
under conditions that are well known in the art. The cleavage products are 
then analyzed by gel electrophoresis as in DNA sequencing. Accordingly, 
this MutY protein is useful in a method for the specific detection and 
localization of A-G mispairs, according to the practice of the present 
invention. 
The full novelty and utility of the present invention may be further 
appreciated by reference to the following brief description of selected 
specific embodiments which advantageously employ various preferred forms 
of the invention as applied to a common problem in genetic mapping of 
point mutations in the human genome. In the course of constructing gene 
linkage maps, for example, it is frequently desirable to compare the 
sequence of a DNA cloned fragment comprising twenty or more kbp of unknown 
sequence (except, perhaps, for a few restriction enzyme recognition sites) 
with homologous sequences in DNA extracted from a human tissue sample. 
While fragments containing sequences homologous to the cloned DNA fragment 
can be detected in the human tissue DNA by the well known "Southern" 
blotting method using radiolabeled DNA of the clone, as explained in the 
Background section, detection and localization of all the sequence 
differences between such a clone and a human DNA sample would be a long 
and arduous task at best using the best methods available in the prior 
art, including restriction enzyme mapping and direct DNA sequencing. 
In contrast, substantially all base pairs in the entire homologous sequence 
of the cloned DNA fragment are compared to those of the human tissue DNA, 
most advantageously in a single test according to the present invention, 
merely by contacting both strands of the human tissue DNA molecule with 
both radiolabeled complementary strands of the second DNA molecule 
(usually without separation from the cloning vector DNA) under conditions 
such that base pairing occurs, contacting the resulting DNA duplexes with 
the E. coli MutS protein that recognizes substantially all base pair 
mismatches under conditions such that the protein forms specific complexes 
with its cognate mispairs, and detecting the resulting DNA:protein 
complexes by contacting the complexes with a membranous nitrocellulose 
filter under conditions such that protein:DNA complexes are retained while 
DNA not complexed with protein is not retained, and measuring the amount 
of DNA in the retained complexes by standard radiological methods. 
If the above detection test indicates the presence of sequence differences 
between the human tissue DNA and the cloned DNA and localization is 
required, or, in the alternative, if such differences are suspected and 
localization as well as detection of them is desired in a first analysis, 
the another method of this invention may be applied for these purposes. An 
embodiment of this aspect of the invention that may be most advantageously 
employed comprises the steps of contacting both strands of the human 
tissue DNA molecule with both radiolabeled complementary strands of the 
second DNA molecule (usually without separation from the cloning vector 
DNA) under conditions such that base pairing occurs, contacting the 
resulting DNA duplexes with a modified form of MutS protein of E. coli to 
which is attached an hydroxyl radical cleaving function under conditions 
such that the radical cleaving function cleaves both strands of the DNA 
within about 20 base pairs of substantially all DNA base mispairs. In the 
absence of any DNA base mispairs in the DNA duplexes comprising 
complementary strands of the human tissue and cloned DNAs, no DNA 
fragments smaller than the cloned DNA (plus vector DNA, if still attached) 
would be detected. Determination of the location of any double-stranded 
DNA cleavages by the modified MutS protein to within a few kbp or less of 
some restriction enzyme cleavage site within the cloned DNA is determined 
by standard restriction enzyme mapping approaches. If greater precision in 
localization and identification of a single base difference is desired, 
sequencing could be confined to those particular fragments of cloned DNA 
that span at least one base sequence difference localized by this method 
and are cleaved by a restriction enzyme at the most convenient distance of 
those sequence differences for direct sequencing. 
The following Examples are provided for further illustrating various 
aspects and embodiments of the present invention and are in no way 
intended to be limiting of the scope. 
EXAMPLE 1 
DNA Mismatch Correction in a Defined System 
In order to address the biochemistry of methyl-directed mismatch 
correction, the reaction has been assayed in vitro using the type of 
substrate illustrated in FIG. 1. Application of this method to cell-free 
extracts of E. coli (A. -L. Lu, S. Clark, P. Modrich, Proc. Natl. Acad. 
Sci. USA 80, 4639, 1983) confirmed in vivo findings that methyl-directed 
repair requires the products of four mutator genes, mutH, mutL, mutS and 
uvrD (also called mutU), and also demonstrated a requirement for the E. 
coli single-strand DNA binding protein (SSB). The dependence of in vitro 
correction on mutH, mutL, and mutS gene products has permitted isolation 
of these proteins in near homogeneous, biologically active forms. The 
97-kD MutS protein binds to mismatched DNA base pairs; the 70-kD MutL 
protein binds to the MutS-Heteroduplex complex (M. Grilley, K. M. Welsh, 
S. -S. Su, P. Modrich, J. Biol. Chem. 264, 1000, 1989); and the 25-kD MutH 
protein possesses a latent endonuclease that incises the unmethylated 
strand of a hemimethylated d(GATC) site (K. M. Welsh, A. -L. Lu, S. Clark, 
P. Modrich, J. Biol. Chem. 262, 15624, 1987), with activation of this 
activity depending on interaction of MutS and MutL with a heteroduplex in 
the presence of ATP (P. Modrich, J. Biol. Chem. 264, 6597, 1989). However, 
these three Mut proteins together with SSB and the DNA helicase II product 
of the uvrD (mutU) gene (I. D. Hickson, H. M. Arthur, D. Bramhill, P. T. 
Emmerson, Mol. Gen. Genet. 190, 265, 1983) are not sufficient to mediate 
methyl-directed repair. Below is described identification of the remaining 
required components and reconstitution of the reaction in a defined 
system. 
Protein and cofactor requirements for mismatch correction. Methyl-directed 
mismatch correction occurs by an excision repair reaction in which as much 
as several kilobases of the unmethylated DNA strand is excised and 
resynthesized (A. -L. Lu, K. Welsh, S. Clark, S. -S. Su, P. Modrich, Cold 
Spring Harbor Symp. Quant. Biol. 49, 589, 1984). DNA polymerase I, an 
enzyme that functions in a number of DNA repair pathways, does not 
contribute in a major way to methyl-directed correction since extracts 
from a polA deletion strain exhibit normal levels of activity. However 
extracts derived from a dnaZ.sup.ts strain are temperature sensitive for 
methyl-directed repair in vitro (Table 1). 
TABLE 1 
______________________________________ 
Requirement for .tau. and .gamma. Subunits of 
DNA Polymerase III Holoenzyme in Mismatch Repair 
Mismatch Correction 
DNA Pol III (fmol/h/mg) Activity 
Extract addition Extract preincubation 
ratio 
genotype 
(ng) 42.degree. 
34.degree. 
42.degree./34.degree.) 
______________________________________ 
dnaZ.sup.ts 
-- 8 91 0.09 
57 ng 75 160 0.47 
dnaZ.sup.+ 
-- 150 160 0.94 
57 ng 160 160 1.0 
______________________________________ 
Extracts from strains AX727 (lac thi str.sup.R dnaZ2016) and AX729 (as 
AX727 except purE dnaz.sup.+) were prepared as described (A. L. Lu, S. 
Clark, P. Modrich, Proc. Natl. Acad. Sci. USA 80, 4639, 1983). Samples 
(110 .mu.g of protein) were mixed with 0.8 .mu.l of 1M KCl and water to 
yield a volume of 7.2 .mu.l, and preincubated at 42.degree. or 34.degree. 
C. for 2.5 minutes. All heated samples were then placed at 34.degree. C. 
and supplemented with 2.2 .mu.l of a solution containi ng 0.1 .mu. g (24 
fmol) of hemimethylated GT heteroduplex DNA, 16 ng of MutL protein, 50 ng 
of MutS protein, and buffer and nucleotide components of the mismatch 
correction assay (A. L. Lu, S. Clark, P. Modrich, Proc. Natl. Acad. Sci. 
USA 80, 4639, 1983). DNA polymerase III holoenzyme (57 ng in 0.6 .mu.l) o 
enzyme buffer was then added, and incubation at 34.degree. C. was 
continued for 60 min. Heated extracts were supplemented with purified Mut 
and MutS proteins because these components a re labile at 42.degree. C. 
Activity measurements reflect the correction of heteroduplex sites. 
The dnaZ gene encodes the T and y subunits of DNA polymerase III holoenzyme 
(M. Kodaira, S. B. Biswas, A. Kornberg, Mol. Gen. Genet. 192, 80, 1983; D. 
A. Mullin, C. L. Woldringh, J. M. Henson, J. R. Walker, Mol. Gen. Genet. 
192, 73, 1983), and mismatch correction activity is largely restored to 
heated extracts of the temperature-sensitive mutant strain by addition of 
purified polymerase III holoenzyme. Since DNA polymerase III holoenzyme is 
highly processive, incorporating thousands of nucleotides per DNA binding 
event, the involvement of this activity is consistent with the large 
repair tracts associated with the methyl-directed reaction. 
Additional data indicate that purified MutH, MutL, and MutS proteins, DNA 
helicase II, SSB, and DNA polymerase III holoenzyme support 
methyl-directed mismatch correction, but this reaction is inhibited by DNA 
ligase, an enzyme that is shown below to be required to restore covalent 
continuity to the repaired strand. This observation led to isolation of a 
55-kD stimulatory protein that obviates ligase inhibition. The molecular 
weight and N-terminal sequence of this protein indicated identity to 
exonuclease I (G. J. Phillips and S. R. Kushner, J. Biol. Chem. 262, 455, 
1987), and homogeneous exonuclease I readily substitutes for the 55-kD 
stimulatory activity (Table 2). Thus, exonuclease I and the six activities 
mentioned above mediate efficient methyl-directed mismatch correction in 
the presence of ligase to yield product molecules in which both DNA 
strands are covalently continuous. 
TABLE 2 
______________________________________ 
Stimulation of in vitro Methyl- 
Directed Correction by Exonuclease I. 
Mismatch correction 
Protein added (fmol/20 min) 
______________________________________ 
None 1 
55-kD protein 18 
Exonuclease I 18 
______________________________________ 
Reactions (10 .mu.l) contained 0.05M HEPES (potassium salt, pH 8.0), 0.02 
KCl, 6 mM MgCl.sub.2, bovine serum albumin (0.05 mg/ml), 1 mM 
dithiothreitol, 2 mM ATP, 100 .mu.M (each) dATP, dCTP, dGTP, and dTTP, 25 
.mu.M .beta.-AND.sup.+, 0.1 .mu.g of hemimethylated, covalently closed GT 
heteroduplex DNA (FIG. 1, methylation on c strand, 24 fmol), 0.26 ng of 
MutH (K. M. Welsh, A. L. Lu, S. Clark, P. Modrich, J. Biol. Chem. 262, 
15624, 1987), 17 ng of MutL (M. Grilley, K. M. Welsh, S. -S. Su, P. 
Modrich, J. Biol. Chem. 264, 1000, 1989), 35 ng of MutS (S. S. Su and P. 
Modrich, Proc. Natl. Acad. Sci. USA 83, 5057, 1986), 200 ng of SSB (T. M. 
Lohman, J. M. Green, R. S. Beyer, Biochemistry 25, 21, 1986; U.S. 
Biochemical Corp.), 10 ng of DNA helicase II (K. Kumura and M. Sekiguchi, 
J. Biol. Chem. 259, 1560, 1984), 20 ng of E. coli DNA ligase (U.S. 
Biochemical Corp.), 95 ng of DNA polymerase III holoenzyme (C. McHenry an 
A. Kornberg, J. Biol. Chem. 252, 6478, 1977), a nd 1 ng of 55kD protein o 
exonuclease I (U.S. Biochemical Corp.) as indicated. Reactions were 
incubated at 37.degree. C. for 20 minutes, quenched at 55.degree. C. for 
10 minutes, chilled on ice, and then digested with Xho I or Hind III 
endonuclease to monitor correction. Repair of the GT mismatch yielded a 
only the GC containing, Xho Isensitive product. 
The requirements for repair of a covalently closed G-T heteroduplex (FIG. 
1) are summarized in Table 3 (Closed circular). No detectable repair was 
observed in the absence of MutH, MutL, or MutS proteins or in the absence 
of DNA polymerase III holoenzyme, and omission of SSB or exonuclease I 
reduced activity by 85 to 90 per cent. 
TABLE 3 
______________________________________ 
Protein and Cofactor Requirements for 
Mismatch Correction in a Defined System. 
Mismatch correction 
(fmol/20 min) 
Closed Circular 
Open Circular 
Reaction conditions 
Heteroduplex 
Heteroduplex 
______________________________________ 
Complete 15 17 (No MutH, No 
ligase) 
minus MutH &lt;1 -- 
minus MutL &lt;1 &lt;1 
minus MutS &lt;1 &lt;1 
minus DNA polymerase 
&lt;1 &lt;1 
III holoenzyme 
minus SSB 2 1.4 
minus exonuclease I 
2 &lt;1 
minus DNA helicase II 
16 15 
minus helicase II, 
&lt;1 &lt;1 
plus immune serum 
minus helicase II, 
14 NT 
plus pre-immune serum 
minus Ligase/AND.sup.+ 
14 -- 
minus MgCl.sub.2 
&lt;1 NT 
minus ATP &lt;1 NT 
minus dNTP's &lt;1 NT 
______________________________________ 
Reactions utilizing covalently closed GT heteroduplex (modification on c 
strand) were performed as described in the legend to TABLE 2 except that 
1.8 ng of exonuclease I was used. Repair of open circular DNA was 
performed in a similar manner except that MutH, DNA ligase, and 
.beta.-AND.sup.+ were omitted from all reactions, and the hemimethylated 
GT heteroduplex (modification on c strand) had been incised with MutH 
protein as described in the legend to FIG. 4. When present, rabb it 
antiserum to helicase II or preimmune serum (5 .mu.g protein) was 
incubated at 0.degree. C. for 20 minutes with reaction mixtures lacking 
MgCl.sub.2 ; the cofactor was then added and the assay was performed as 
above. Although not shown, antiserum inhibition was reversed by the 
subsequent addition of more helicase II. With the exception of the DNA 
polymerase III preparation, which contained about 15% by weight DNA 
helicase II (text), the purity of individual protein fractions was 
.gtoreq.95% . NT - not tested. 
These findings are in accord with previous conclusions concerning 
requirements of the methyl-directed reaction. However, in contrast to 
observations in vivo and in crude extracts indicating a requirement for 
the uvrD product, the reconstituted reaction proceeded readily in the 
absence of the added DNA helicase II (Table 2). Nevertheless, the reaction 
was abolished by antiserum to homogeneous helicase II, suggesting a 
requirement for this activity and that it might be present as a 
contaminant in one of the other proteins. Analysis of these preparations 
for their ability to restore mismatch repair to an extract derived from a 
uvrD (mutU) mutant and for the physical presence of helicase II by 
immunoblot assay revealed that the DNA polymerase III holoenzyme 
preparation contained sufficient helicase II (13 to 15 per cent of total 
protein by weight) to account for the levels of mismatch correction 
observed in the defined system. Similar results were obtained with 
holoenzyme preparations obtained from two other laboratories. The purified 
system therefore requires all the proteins that have been previously 
implicated in methyl-directed repair. 
The rate of correction of the closed circular heteroduplex was unaffected 
by omission of DNA ligase (Table 3), but the presence of this activity 
results in production of a covalently closed product. Incubation of a 
hemimethylated, supercoiled G-T heteroduplex with all seven proteins 
required for correction in the presence of DNA ligase resulted in 
extensive formation of covalently closed, relaxed, circular molecules. 
Production of the relaxed DNA was dependent on MutS (FIG. 2) and MutL 
proteins, and the generation of this species was associated with 
heteroduplex repair (FIG. 2). Correction also occurred in the absence of 
ligase, but in this case repair products were open circular molecules, the 
formation of which depended on the presence of MutS (FIG. 2). Since MutS 
has no known endonuclease activity but does recognize mispairs, it is 
inferred that open circular molecules are the immediate product of a 
mismatch-provoked excision repair process. Ligase closure of the strand 
break(s) present in this species would yield the covalently closed, 
relaxed circular product observed with the complete system. 
The set of purified activities identified here as being important in 
methyl-directed repair support efficient correction. In the experiments 
summarized in Table 3, the individual proteins were used at the 
concentrations estimated to be present in the standard crude extract assay 
for correction as calculated from known specific activity determinations. 
Under such conditions the rate and extent of mismatch repair in the 
purified system are essentially identical to those observed in cell-free 
extracts. 
DNA sites involved in repair by the purified system. The single d(GATC) 
sequence within the G-T heteroduplex shown in FIG. 1 is located 1024 base 
pairs from the mispair. Despite the distance separating these two sites, 
correction of the mismatch by the purified system responded to the state 
of modification of the d(GATC) sequence as well as its presence within the 
heteroduplex (FIG. 3). A substrate bearing d(GATC) methylation on both DNA 
strands did not support mismatch repair nor did a related heteroduplex in 
which the d(GATC) sequence was replaced by d(GATT). However, each of the 
two hemimethylated heteroduplexes were subject to strand-specific 
correction, with repair in each case being restricted to the unmodified 
DNA strand. With a heteroduplex in which neither strand was methylated, 
some molecules were corrected on one strand, and some were corrected on 
the other. As can be seen, the hemimethylated heteroduplex bearing 
methylation on the complementary DNA strand was a better substrate than 
the alternative configuration in which modification was on the viral 
strand, with a similar preference for repair of the viral strand being 
evident with the substrate that was unmethylated on either strand. This 
set of responses of the purified system to the presence and state of 
modification of d(GATC) sites reproduce effects previously documented in 
vivo and in crude extract experiments (R. S. Lahue, S. -S. Su, P. Modrich, 
Proc. Natl. Acad. Sci. USA 84, 1482, 1987). 
The efficiency of repair by the methyl-directed pathway depends not only on 
the nature of the mispair, but also on the sequence environment in which 
the mismatch is embedded (P. Modrich, Ann. Rev. Biochem. 56, 435, 1987). 
To assess the mismatch specificity of the purified system under conditions 
where sequence effects are minimized, a set of heteroduplexes were used in 
which the location and immediate sequence environment of each mispair are 
essentially identical (S. -S. Su, R. S. Lahue, K. G. Au, P. Modrich, J. 
Biol. Chem. 263, 6829, 1988). This analysis (Table 4) showed that the 
TABLE 4 
______________________________________ 
Correction Efficiencies for Different Mismatches. 
Methylation State 
C.sup.+ V.sup.- 
C.sup.- V.sup.+ 
Heteroduplex Markers Rate Bias Rate Bias 
______________________________________ 
C 5'-CTCGA G AGCTT 
Xho I 1.2 &gt;18 0.38 &gt;5 
V 3'-GAGCT T TCGAA 
Hind III 
C 5'-CTCGA G AGCTG 
Xho I 1.1 &gt;17 0.38 &gt;6 
V 3'-GAGCT G TCGAC 
Pvu II 
C 5'-ATCGA T AGCTT 
Cla I 1.0 &gt;16 0.24 3 
V 3'-TAGCT T TCGAA 
Hind III 
C 5'-ATCGA A AGCTT 
Hind III 0.88 &gt;20 0.20 &gt;7 
V 3'-TAGCT A TCGAA 
Cla I 
C 5'-CTCGA A AGCTT 
Hind III 0.61 17 0.28 &gt;5 
V 3'-GAGCT C TCGAA 
Xho I 
C 5'-GTCGA C AGCTT 
Sal I 0.60 12 0.23 &gt;4 
V 3'-CAGCT T TCGAA 
Hind III 
C 5'-GTCGA A AGCTT 
Hind III 0.44 &gt;13 0.21 5 
V 3'-CAGCT G TCGAA 
Sal I 
C 5'-CTCGA C AGCTG 
Pvu II 0.04 NS &lt;0.04 NS 
V 3'-GAGCT C TCGAC 
Xho I 
______________________________________ 
Correction of the eight possible basebase mispairs was tested with the se 
of covalently closed heteroduplexes described previously including the GT 
substrate shown in FIG. 1. With the exception of the mispair and the 
variations shown at the fifth position on either side, all heteroduplexes 
were identical in sequence. Each DNA was tested in both hemimethylated 
configurations under complete reaction conditions (Table 3, closed 
circular heteroduplex) except that samples were removed at 5-minute 
intervals over a 20 minute period in order to obtain initial rates 
(fmol/min). c and v refer to complementary and viral DNA strands, and Bia 
indicates the relative efficiency of mismatch repair occurring on the two 
DNA strands (ratio of unmethylated to methylated) as determined 60 minute 
after the reaction was started. NS not significant. With the exception o 
the CC heteroduplexes, repair in the absence of MutS protein was less tha 
20% (in most cases &lt;10%) of that obser ved in its presence (not shown). 
purified system is able to recognize and repair in a methyl-directed manner 
seven of the eight possible base-base mismatches, with C-C being the only 
mispair that was not subject to significant correction. Table 3 also shows 
that the seven corrected mismatches were not repaired with equal 
efficiency and that in the case of each heteroduplex, the hemimethylated 
configuration modified on the complementary DNA strand was a better 
substrate than the other configuration in which the methyl group was on 
the viral strand. These findings are in good agreement with patterns of 
repair observed with this set of heteroduplexes in E. coli extracts 
(Although the patterns of substrate activity observed in extracts and in 
the purified system are qualitatively identical, the magnitude of 
variation observed differs for the two systems. Hemimethylated 
heteroduplexes modified on the complementary DNA strand are better 
substrates in both systems, but in extracts such molecules are repaired at 
about twice the rate of molecules methylated on the viral strand. In the 
purified system these relative rates differ by factors of 2 to 4. A 
similar effect may also exist with respect to mismatch preference within a 
given hemimethylated family. Although neither system repairs C-C, the 
rates of repair of other mismatches vary by a factors of 1.5 to 2 in 
extracts but by factors of 2 to 3 in the defined system.). 
Strand-specific repair directed by a DNA strand break. Early experiments on 
methyl-directed repair in E. coli extracts led to the proposal that the 
strand-specificity of the reaction resulted from endonucleolytic incision 
of an unmethylated DNA strand at a d(GATC) sequence. This idea was 
supported by the finding that purified MutH protein has an associated, but 
extremely weak d(GATC) endonuclease that is activated in a 
mismatch-dependent manner in a reaction requiring MutL, MutS, and ATP. The 
purified system has been used to explore this effect more completely. 
The two hemimethylated forms of the G-T heteroduplex shown in FIG. 1 were 
incised using high concentrations of purified MutH protein to cleave the 
unmethylated DNA strand at the d(GATC) sequence (&gt;&gt;pGpApTpC). After 
removal of the protein, these open circular heteroduplexes were tested as 
substrates for the purified system in the absence of DNA ligase. Both open 
circular species were corrected in a strand-specific manner and at rates 
similar to those for the corresponding covalently closed heteroduplexes 
(FIG. 4). As observed with closed circular heteroduplexes, repair of the 
MutH-cleaved molecules required MutL, MutS, SSB, DNA polymerase III 
holoenzyme, and DNA helicase II (FIG. 4 and open circle entries of Table 
2), but in contrast to the behavior of the closed circular substrates, 
repair of the mismatch within the open circular molecules occurred readily 
in the absence of MutH protein. Thus prior incision of the unmethylated 
strand of a d(GATC) site can bypass the requirement for MutH protein in 
strand-specific mismatch correction. 
The nature of the MutH-independent repair was examined further to assess 
the effect of ligase on the reaction and to determine whether a strand 
break at a sequence other than d(GATC) can direct correction in the 
absence of MutH protein (FIG. 5). As mentioned above, a covalently closed 
G-T heteroduplex that lacks a d(GATC) sequence is not subject to repair by 
the purified system in the presence (FIG. 3) or absence of DNA ligase. 
However, the presence of one strand-specific, site-specific break is 
sufficient to render this heteroduplex a substrate for the purified system 
in the absence of ligase and MutH protein (FIG. 5). Repair of this open 
circular heteroduplex was limited to the incised, complementary DNA 
strand, required presence of MutL and MutS proteins, DNA polymerase III, 
and SSB, and correction of the molecule was as efficient as that observed 
with the hemimethylated heteroduplex that had been cleaved by MutH at the 
d(GATC) sequence within the complementary strand. Although the presence of 
a strand break is sufficient to permit strand-specific correction of a 
heteroduplex in the absence of MutH and ligase, the presence of the latter 
activity inhibited repair not only on the heteroduplex lacking a d(GATC) 
sequence but also on both hemimethylated molecules that had been 
previously incised with MutH protein (FIG. 5). This inhibition by ligase 
was circumvented by the presence of MutH protein, but only if the 
substrate contained a d(GATC) sequence, with this effect being 
demonstrable when both types of heteroduplex were present in the same 
reaction (FIG. 5, last column). This finding proves that MutH protein 
recognizes d(GATC) sites and is consistent with the view that the function 
of this protein in mismatch correction is the incision of the unmethylated 
strand at this sequence. 
EXAMPLE 2 
Purification of MutY Protein 
Purification of MutY Protein. E. coli RK1517 was grown at 37.degree. C. in 
170 liters of L broth containing 2.5 mM KH.sub.2 PO.sub.4, 7.5 mM Na.sub.2 
HPO.sub.4 (culture pH=7.4) and 1% glucose. The culture was grown to an 
A590 of 4, chilled to 10.degree. C. and cells were harvested by continuous 
flow centrifugation. Cell paste was stored at -70.degree. C. A summary of 
the MutY purification is presented in Table 1. Fractionation procedures 
were performed at 0.degree.-4.degree. C., centrifugation was at 
13,000.times.g, and glycerol concentrations are expressed as volume 
percent. 
Frozen cell paste (290 g) was thawed at 4.degree. C., resuspended in 900 ml 
of 0.05M Tris-HCl (pH 7.5), 0.1M NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 
and cells were disrupted by sonication. After clarification by 
centrifugation for 1 hr, the lysate (Fraction I, 970 ml) was treated with 
185 ml of 25% streptomycin sulfate (wt/vol in 0.05M Tris-HCl (pH 7.5), 
0.1M NaCl, 1 mM dithiothreitol, 0.1 mM EDTA) which was added slowly with 
stirring. After 30 min of additional stirring, the solution was 
centrifuged for 1 h, and the supernatant (1120 ml) was treated with 252 g 
of solid ammonium sulfate which was added slowly with stirring. After 30 
min of additional stirring, the precipitate was collected by 
centrifugation for 1 h, resuspended to a final volume of 41 ml in 0.02M 
potassium phosphate (pH 7.5), 0.1 mM EDTA, 10% (vol/vol) glycerol, 1 mM 
dithiothreitol, and dialyzed against two 2 l portions of 0.02M potassium 
phosphate (pH 7.5), 0.1M KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10% 
glycerol (2 h per change). The dialyzed material was clarified by 
centrifugation for 10 min to yield Fraction II (45 ml). 
Fraction II was diluted 10-fold into 0.02M potassium phosphate (pH 7.5), 
0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol so that the conductivity of 
the diluted solution was comparable to that of the dilution buffer 
containing 0.1M KCl. The dilution was performed on small aliquots of 
Fraction II, and diluted samples were immediately loaded at 1 ml/min onto 
a 14.7 cm.times.12.6 cm.sup.2 phosphocellulose column equilibrated with 
0.02M potassium phosphate (pH 7.5), 0.1M KCl, 0.1 mM EDTA, 1 mM 
dithiothreitol, 10% glycerol. The column was washed with 400 ml of 
equilibration buffer, and developed with a 2 liter linear gradient of KCl 
(0.1 to 1.0M) in 0.02M potassium phosphate (pH 7.5), 0.1 mM EDTA, 1 mM 
dithiothreitol, 10% glycerol. Fractions containing MutY activity, which 
eluted at about 0.4M KCl, were pooled (Fraction III, 169 ml). 
Fraction III was dialyzed against three 2 liter portions of 5 mM potassium 
phosphate (pH 7.5), 0.05M KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10% 
glycerol (2 h per change) until the conductivity was comparable to that of 
the dialysis buffer. After clarification by centrifugation at for 10 min, 
the solution was loaded at 0.5 ml/min onto a 21 cm.times.2.84 cm.sup.2 
hydroxylapatite column equilibrated with 5 mM potassium phosphate, pH 7.5, 
0.05M KCl, 1 mM dithiothreitol, 10% glycerol. After washing with 130 ml of 
equilibration buffer, the column was eluted with a 600 ml linear gradient 
of potassium phosphate (5 mM to 0.4M, pH 7.5) containing 0.05M KCl, 1 mM 
dithiothreitol, 10% glycerol. Fractions eluting from the column were 
supplemented with EDTA to 0.1 mM. Peak fractions containing 60% of the 
total recovered activity, which eluted at about 0.1M potassium phosphate, 
were pooled (Fraction IV, 24 ml). The remaining side fractions contained 
impurities which could not be resolved from MutY by MonoS chromatography. 
Fraction IV was diluted by addition of an equal volume of 0.05M KCl, 0.1 mM 
EDTA, 1 mM dithiothreitol, 10% glycerol. After clarification by 
centrifugation for 15 min, diluted Fraction IV was loaded at 0.75 ml/min 
onto a Pharmacia HR 5/5 MonoS FPLC column that was equilibrated with 0.05M 
sodium phosphate (pH 7.5), 0.1M NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 
10% glycerol. The column was washed at 0.5 ml/min with 17 ml of 
equilibration buffer and developed at 0.5 ml/min with a 
TABLE 1 
______________________________________ 
Purification of MutY protein from 290 g of E. coli RK1517 
Specific 
Frac- Total Protein 
Activity 
Yield 
tion Step mg units/mg 
Percent 
______________________________________ 
I Extract 10,900 40 (100) 
II Ammonium sulfate 
1,350 272 84 
III Phosphocellulose 
66 10,800 160 
IV Hydroxylapatite 
1.4 136,000 
44 
V MonoS 0.16 480,000 
18 
______________________________________ 
Specific A.G to CG mismatch correction in cellfree extracts was determine 
as described previously (Au et al. 1988), except that ATP and glutathione 
were omitted from the reaction and incubation was for 30 min instead of 1 
h. For complementation assays, each 0.01 ml reaction contained RK1517Y33 
extract (mutS mutY) at a concentration of 10 mg/ml protein. One unit of 
MutY activity is defined as the amount required to convert 1 fmol of A.G 
mismatch to CG base pair per h under compleme ntation conditions. 
20 ml linear gradient of NaCl (0.1 to 0.4M) in 0.05M sodium phosphate (pH 
7.5), 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol. Fractions with 
MutY activity, which eluted at approximately 0.2M NaCl, were pooled 
(Fraction V, 2.6 ml). Fraction V was divided into small aliquots and 
stored at -70.degree. C. 
Assay for MutY-dependent, A.G-specific glycosylase 
DNA restriction fragments were labeled at either the 3' or 5' ends with 
.sup.32 P. Glycosylase activity was then determined in 0.01 ml reactions 
containing 10 ng end-labeled DNA fragments, 0.02M Tris-HCl, pH 7.6, 1 mM 
EDTA, 0.05 mg/ml bovine serum albumin, and 2.7 ng MutY. After incubation 
at 37.degree. C. for 30 min, the reaction mixture was treated with 
2.5.times.10.sup.-3 units of HeLa AP endonuclease II in the presence of 11 
mM MgCl.sub.2 and 0.005% Triton X-100 for 10 min at 37.degree. C. 
Reactions were quenched by the addition of an equal volume of 80% 
formamide, 0.025% xylene cyanol, 0.025% bromphenol blue, heated to 
80.degree. C. for 2 min, and the products analyzed on an 8% sequencing 
gel. Control reactions contained either no MutY, no A.G mismatch or no AP 
endonuclease II. 
Strand cleavage at the AP site generated by MutY could also be accomplished 
by treatment with piperidine instead of treatment with AP endonuclease II. 
After incubation for 30 min. at 37.degree. C. with MutY as described 
above, the reaction mixture was precipitated with ethanol in the presence 
of carrier tRNA, then resuspended in 1M piperidine and heated at 
90.degree. C. for 30 min. After two additional ethanol precipitations, 
changing tubes each time, the pellet was resuspended in a minimum volume 
of water to which was added an equal volume of 80% formamide, 0.025% 
xylene cyanol, 0.025% bromphenol blue. The products were then analyzed on 
an 8% sequencing gel. 
For purposes of completing the background description and present 
disclosure, each of the published articles, patents and patent 
applications heretofore identified in this specification are hereby 
incorporated by reference into the specification. 
The foregoing invention has been described in some detail for purposes of 
clarity and understanding. It will also be obvious that various 
combinations in form and detail can be made without departing from the 
scope of the invention.