Accumulation of the Cyclobutane Thyminie Dime in Defined 

Sequences of Free and Nucleosomal DNA

by Amethist S. Finch, William B. Davis, and Steven E. Rokita

ARL-RP-0455 August 2013

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ARL-RP-0455 August 2013

Accumulation of the Cyclobutane Thyminie Dime in Defined 

Sequences of Free and Nucleosomal DNA

Amethist S. Finch, William B. Davis, and Steven E. Rokita

Sensors and Electron Devices Directorate, ARL

A reprint from the Photochem Photobiol, Sci, 2013.

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Accumulation of the Cyclobutane Thyminie Dime in Defined Sequences of Free 

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14. ABSTRACT

Photochemical cyclobutane dimerization of adjacent thymines generates the major lesion in DNA caused by exposure to 

sunlight. Not all nucleotide sequences and structures are equally susceptible to this reaction or its potential to create mutations. 

Photostationary levels of the cyclobutane thymine dimer have now been quantified in homogenous samples of DNA 

reconstituted into nucleosome core particles to examine the basis for previous observations that such structures could induce a 

periodicity in dimer yield when libraries of heterogeneous sequences were used. Initial rate studies did not reveal a similar 

periodicity when a homogenous core particle was analyzed, but this approach examined only formation of this

photochemically reversible cyclobutane dimer. Photostationary levels result from competition between dimerization and 

reversion and, as described in this study, still express none of the periodicity within two alternative core particles that was 

evident in heterogeneous samples. Such periodicity likely arises from only a limited set of sequences and structural 

environments that are not present in the homogeneous and well-characterized assemblies available to date.

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Photochemical &

Photobiological Sciences

PAPER

Cite this: Photochem. Photobiol. Sci., 2013,

12, 1474

Received 14th May 2013,

Accepted 14th June 2013

DOI: 10.1039/c3pp50147g

www.rsc.org/pps

Accumulation of the cyclobutane thymine dimer in

defined sequences of free and nucleosomal DNA†

Amethist S. Finch,‡a William B. Davisb and Steven E. Rokita*§a

Photochemical cyclobutane dimerization of adjacent thymines generates the major lesion in DNA caused

by exposure to sunlight. Not all nucleotide sequences and structures are equally susceptible to this reaction or its potential to create mutations. Photostationary levels of the cyclobutane thymine dimer have

now been quantified in homogenous samples of DNA reconstituted into nucleosome core particles to

examine the basis for previous observations that such structures could induce a periodicity in dimer yield

when libraries of heterogeneous sequences were used. Initial rate studies did not reveal a similar periodicity when a homogenous core particle was analyzed, but this approach examined only formation of this

photochemically reversible cyclobutane dimer. Photostationary levels result from competition between

dimerization and reversion and, as described in this study, still express none of the periodicity within two

alternative core particles that was evident in heterogeneous samples. Such periodicity likely arises from

only a limited set of sequences and structural environments that are not present in the homogeneous

and well-characterized assemblies available to date.

Introduction

Covalent dimerization of adjacent thymines through a photochemical [2 + 2] reaction was first described over 50 years ago.1

The resulting thymine–thymine cyclobutane dimer product

was soon identified as the major DNA lesion formed by UV

irradiation of cells and was associated with the mutagenic and

carcinogenic effects of solar irradiation.2,3 However, direct connection between thymine dimerization and photomutation is

complicated by many factors including those controlling

dimer accumulation and its repair (Scheme 1). Both the distribution of cyclobutane thymine dimers and the genetic

changes caused by irradiation are heterogeneous within cellular DNA. A purely statistical correlation between frequency of

dimerization, number of adjacent thymines, and mutation rate

is not often observed. Instead, dimer formation and repair

variably contribute to the mutational profiles. Each process is

affected by nucleotide sequence, conformation and higher

ordered structures.4–9 The biological consequences of a cyclobutane dimer within a particular sequence have been investigated by incorporating a dimer generated synthetically into

defined polynucleotides.10–13 Likewise, relative efficiencies of

thymine dimerization have been examined for sequence and

Scheme 1 Formation and repair of the cyclobutane thymine dimer.

†Electronic supplementary information (ESI) available: Fig. S1, denaturing polyacrylamide gel analysis of thymine dimerization within the 601 sequence alone

and reconstituted into a nucleosome core particle; Fig. S2, photoproduct detection using the 3′–5′ exonuclease activity of T4 polymerase; Fig. S3, approach to a

photostationary level of thymine dimer from duplex DNA alternatively containing a single thymine pair or its dimer analog. See DOI: 10.1039/c3pp50147g

‡Current address: US Army Research Laboratory, 2800 Powder Mill Road,

Adelphi, MD 20783, USA.

§Current address: Department of Chemistry, Johns Hopkins University,

3400 N. Charles St., Baltimore, MD 21218, USA. E-mail: Rokita@jhu.edu

a

Department of Chemistry and Biochemistry, University of Maryland, College Park,

MD 20742, USA. E-mail: Rokita@jhu.edu

b

School of Molecular Biosciences, Washington State University, Pullman, WA 99164,

USA

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structure dependence using oligo- and polynucleotides of

defined sequence.

The effect of neighboring sequences on thymine dimerization was evaluated in a number of laboratories by determining

its initial rate of formation within various gene fragments.

Although a consensus has not been achieved, thymine dimers

are generally more prevalent when flanked by pyrimidines and

adenines and less common when flanked by guanine.14–17

Single-stranded DNA also generates more cyclobutane thymine

dimers than double-stranded DNA of equivalent sequence,18

and triplex formation further suppresses thymine dimerization

in a helix.19,20 Along tracts of (dT)n/(dA)n, dimers seem to

accumulate at the 3′-terminal dithymine when irradiated as a

duplex in contrast to a uniform distribution of dimers that

forms from single-stranded poly(dT).21 Photodimerization

within heterogeneous sequences of DNA wrapped in nucleosome core particles (NCP) results in a fascinating periodicity of

10.3 bases and suggest maximum yields of these lesions are

generated at sites for which the phosphodiester backbone is

farthest from the histones.22–24 A similar distribution of cyclobutane dimers is not evident after irradiation of the same DNA

in the absence of NCP. The periodicity of reaction in NCP has

been attributed to the differential curvature of its DNA, and a

similar periodicity has been observed after a heterogeneous

collection of DNA duplexes was constrained by looping

enforced by association of Lambda repressor.25

DNA dynamics, conformation and flexibility have all been

implicated in the yield of cyclobutane thymine dimerization.

For example, dimerization was suppressed near the dyad axis

of the NCP, a region known to perturb the helical nature of

duplex DNA.22 Model oligonucleotides variably describe the

importance of context and suggest that particular conformers

may be predisposed to efficient photochemical dimerization.26

Excited-state properties may equally control reaction by

affecting the lifetime and population of electronic states

required for dimerization.27–30 These properties are similarly

dependent on sequence as are subsequent delocalization of

excited states through energy and charge transfer.31–35 From a

theoretical approach, the helical twist of DNA was proposed as

most critical for dimerization.36

To date, the vast majority of studies have focused on the

initial rate of cyclobutane dimer formation even though this

process is reversible under standard model conditions that

include use of the 254 nm emission from a low pressure

mercury lamp.37–39 Moreover, the parent thymine pair is

favored over its dimer under photostationary conditions.40 The

net accumulation of dimers should then be influenced by the

rate of dimerization and photoreversion, and together these

may create sites known as hotspots for thymine dimerization.

The efficiency of photoreversion, similar to photodimerization,

is affected by the helical structure and local sequence surrounding the thymine pairs.41,42 This reversion has been

described as a process of self-repair and was first observed at

sites proximal to guanine quadruplexes.43 Electron donors and

acceptors associated with DNA have also demonstrated the

ability to repair thymine dimers.44–46 We have since returned

to the topic of thymine dimerization from the perspective of

its photostationary state, as reported here, to help examine the

origins of its accumulation within nucleosomal DNA.

Experimental section

General materials

Chemicals were purchased from Fisher Scientific (Waltham,

MA) or Sigma-Aldrich (St. Louis, MO) and were enzymatic

grade or the highest grade available. All aqueous solutions

were prepared with purified water (Barnstead NANOpure II

purifier, 17.8 MΩ). Oligonucleotide primers were purified by

polyacrylamide gel electrophoresis (PAGE) and purchased from

IDT DNA (Coralville, IA) and Gibco Bioresearch Labs (Carlsbad,

California). Washed pooled chicken blood (30%) in Alsevers

buffer was purchased from Lampire Biological Laboratories

(Pipersville, PA), frozen in liquid N2, and stored at −80 °C until

use. Plasmid XP10 (pXP 10) containing the 5S RNA gene of

Xenopus borealis was obtained from the late Dr A. Wolffe.

Plasmid GEM-3z (pGEM) containing the 601 positioning

sequence was obtained from the late Dr J. Widom.

General methods

The 154 bp 5S gene was generated by standard PCR (100 μL) of

pXP10 (100 pg) with primers 5′-AAT TCG AGC TCG CCC GGG

GAT CCG-3′ and 5′-ACT AAC CAG GCC CGA CCC TGC TTC-3′

(50 pmol each) and Vent DNA polymerase (New England

Biolabs, Ipswich, MA). Radiolabeled samples were generated

analogously with 5′-[32P]-ACT AAC CAG GCC CGA CCC TGC

TTC-3′. A 162 bp sequence containing the 601 sequence for

nucleosome orientation was similarly generated by PCR of

pGEM with primers NH2-(CH2)6-5′-TAT ACG CGG CCG CCC

TGG-3′ and 5′-CAC AGG ATG TAT ATA TCT GAC AC-3′ as

described previously.47 The presence of an aminolinker at

one 5′-terminus allowed for selective radiolabeling of the other

5′-terminus with γ-[32P]-ATP and T4 polynucleotide kinase

(New England Biolabs) following manufacturer protocols.

Histone isolation and chromatin reconstitution to form the

nucleosome core particles (NCP) followed published

procedures32,48,49 and was confirmed by native PAGE (6%) on a

MiniProtean3 apparatus (Bio-Rad, Hercules, CA).

Irradiation of DNA and detection of the cyclobutane thymine

dimer

DNA alone and reconstituted in the NCPs (200 kdpm) was irradiated in 10 mM potassium phosphate (pH 7.0) and 100 mM

NaCl (10 μL) using a low pressure Hg pencil lamp (6035

Hg-Argon) (Oriel, Stratford, CT). The height of the pen lamp

was varied from 6–11 cm to create a light flux from

0.016–0.16 mW cm−2 as needed. Histones were removed from

NCP after irradiation by standard chloroform-phenol extraction, and DNA was treated with T4 endonuclease V (endo V)

(EpiCentre, Madison, WI) according to manufacturer conditions at 37 °C for 5 h. Samples were analyzed by denaturing

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PAGE (8%) and quantified by phosphoimagery and ImageQuant software v.5.2 (GE Healthcare, Pittsburgh, PA).

Results

The influence of helical structure on the propensity of duplex

DNA to form cyclobutane thymine dimers was perhaps best

demonstrated by the periodicity of its distribution within DNA

bent around the core histones or looped between other DNAbinding proteins.22–25 A detailed correlation between local

sequence, structure and dimer accumulation was not initially

possible since the data represented a composite of reactivity

for heterogeneous sequences bound to the NCP in numerous

translational and rotational orientations. More detailed analysis requires a target of defined sequence that forms a uniform

assembly. This was first accomplished with the relatively small

gene encoding 5S rRNA that forms a NCP of homogeneous

structure (Fig. 1).50 The same gene and its NCP were consequently selected to initiate the current study focusing on the

photostationary levels of thymine dimer.

The 5S gene (X. borealis) was irradiated using a mercury

pen lamp (254 nm), and formation of the cyclobutane thymine

dimer was detected by subsequent fragmentation of the DNA

by T4 endonuclease V, an enzyme specific for this type of pyrimidine dimer.51 The resulting DNA was separated by denaturing polyacrylamide gel electrophoresis, and each fragment was

quantified by phosphoimagery (Fig. 2). As expected, the major

fragments increased with exposure to UV irradiation, and their

gel mobility was consistent with the expected sites of thymine

dimers. Irradiation was continued beyond that used for

measuring initial rates in order to approach the photostationary levels of dimer accumulation. However, further irradiation

degraded the parent DNA and its dimer-containing

fragments non-selectively. DNA was also treated with heat and

piperidine before and after the maximum irradiation time

(30 min) to detect base labile products. The lack of significant

fragmentation of the DNA under these conditions indicated

that nonspecific degradation was not competitive with

thymine dimerization. Analysis of the two sites closest to the 3′

terminus was not included in these investigations. Dimer 1 is

beyond the region wrapped within the NCP, and the fragment

size associated with dimerization at site 2 was obscured by a

fraction of the parent sequence that migrated anomalously

during electrophoresis.

The collective rate of thymine dimerization and the total

accumulation of thymine dimers within the 5S gene did not

significantly vary in the presence or absence of the core histones or when reconstituted to form the NCP (Fig. 3). Under

the experimental conditions, a photostationary level of ca. 0.5

cyclobutane dimer per duplex was established within 20 min

of irradiation. The distribution of dimers among the individual sites was also monitored over time and was not affected by

association of the DNA with the core histones (Fig. 4). Dimer 3

(Fig. 1) formed in lowest yield (ca. 4%) under all conditions

and reached its photostationary state within 5 min. In contrast,

dimer 9 accumulated to a yield of approximately 8% after

20 min. At this position, and to a lesser extent at position 7,

more dimer forms in the NCP than in the free duplex.

Additionally, a photostationary state may not be achieved after

30 min of irradiation, but the scatter in the data limits further

interpretation. Regardless of this constraint, the results are

still not reminiscent of the dramatic effect of the NCP on

Fig. 1 Translational and rotational orientation of the 5S rRNA gene (X. borealis)

within the nucleosome core particle. Nucleobases are closest to the histone

octamer at the minima and farthest at the maxima of the line above the

DNA sequence. Sites of potential thymine dimerization are indicated as (1)

through (10).

Fig. 2 Formation of the cyclobutane thymine dimer within the 5S rRNA gene

(X. borealis). Yields and distribution of the cyclobutane photoproduct were

determined by strand fragmentation using T4 endonuclease V after irradiation,

polyacrylamide gel electrophoresis (8%) and phosphoimage analysis. The radiolabeled 5S gene as the free duplex and reconstituted in a NCP as illustrated in

Fig. 1 were analyzed after the indicated time of irradiation (254 nm). Equivalent

samples before (OP) and after irradiation (30 min) (30P) were heated with piperidine (1 M) for 30 min at 90 °C.

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dimer distribution observed previously for a heterogeneous

array of DNA sequences.22–25

In contrast to the NCP formed collectively by the previous

array of sequences, the NCP formed by the 5S gene alone provided only limited-TT-positions for detecting a possible correlation between duplex structure and photostationary levels of

the cyclobutane thymine dimer. A second NCP based on a

duplex sequence with stronger rotational and translational

positioning than the 5S gene was next investigated to expand

the range of sites available for dimer formation. The DNA used

for this was discovered by the Widom laboratory as “sequence

601” through a systematic selection of strong nucleosome

positioning sequences.52 Again, thymine–thymine sites are

limited, but at least two (sites 2 and 5) represent regions in

which the nucleobases are proximal to the histone octamer,

and two (sites 1 and 6) represent regions in which the nucleobases are relatively distal to the histone octamer (Fig. 5).

The free 601 sequence and its reconstituted NCP were irradiated under the same conditions as described above for the

5S gene. Again, no significant differences in the collective level

of the cyclobutane thymine dimers were detected under photostationary conditions in the presence or absence of the associated histone octamer (Fig. 6). Both yielded ca. 0.4 dimer per

duplex (Fig. 6). The short time needed to establish this yield

versus that needed for the 5S gene was not a function of DNA

sequence but rather the replacement of an aging lamp. As in

the analysis of the 5S gene, the levels of dimer in the 601

sequence did not include data from position 1 due to the lack

of resolution between its fragment formed by the endonuclease and the parent DNA after denaturing gel electrophoresis

(ESI†). Further investigation to measure the dimerization

of thymine at individual sites was not pursued since

reconstitution to the NCP had no apparent effect on its overall

yield. This represents the second example of an individual

DNA sequence that did not replicate the variable formation of

dimer in a heterogeneous population of sequences.

Discussion

The use of defined sequences of DNA was crucial when first

measuring the influence of neighboring nucleobases on the

accumulation of cyclobutane thymine dimers.14–17,41 In contrast, large populations of diverse sequences were necessary to

detect the effects of conformational constraints on dimerization within duplex DNA such as those enforced by association

with proteins.22–25 A previous attempt to identify the interplay

between individual sequences and conformation did not

recapitulate the earlier studies based on the heterogeneous

mixture of sequences. No significant modulation was observed

in the initial rates of thymine dimerization when measured in

the 5S gene alone and after reconstitution with the core

histone proteins.50 However, this kinetic approach focused on

only half of the reversible processes. While dimer formation is

dominated initially by the forward reaction, ultimately a photostationary level of dimer should form that reflects the relative

efficiencies of reversion vs. formation. A second set of

sequence and conformational variables combine to control

thymine dimer reversion, and thus, photostationary levels of

this dimer were determined as an alternative basis for the

periodicity of dimerization in heterogeneous samples.

After irradiation for 10–30 min, the population of cyclobutane dimers at most positions became constant as expected

for a photostationary state (Fig. 3, 4 and 6). However, alternative explanations for the limited yield of dimer were possible.

The data might also have been consistent with full consumption of a highly reactive subpopulation of DNA sequences or

conformers. This possibility was minimized by selecting

systems that form highly uniform NCP.50,52 The duplex DNA in

the presence and absence of protein was also expected to be

sufficiently dynamic over the time of irradiation to equilibrate

between subtle differences in conformation. The accumulation

of undetected photochemical products such as the Dewar and

(6-4) dimers might have additionally suppressed full conversion to cyclobutane dimer, but this too was not likely. The

dominant product generated by irradiation at 254 nm is the

cyclobutane thymine–thymine dimer followed by its related

thymine–cytosine dimer, the thymine–cytosine 6-4 product

and the cyclobutane cytosine–thymine dimer.53 T4 endonuclease V was used to focus on the cyclobutane dimers since

only these are recognized as substrates.54 Once again, the

cyclobutane thymine–thymine dimer dominated the product

profiles observed. Generation of competing products was

checked with a complementary assay based on the 3′–5′ exonuclease activity of T4 DNA polymerase. This approach had

previously been used to detect a broad array of DNA lesions

including the thymine–cytosine 6-4 photoproduct.50,55,56

Again, the major photochemical product was dimerization at

Fig. 3 Time-dependent formation of the cyclobutane thymine dimer in the 5S

rRNA gene. Total incorporation of cyclobutane dimers (T^T) within the 5S gene

sequence was determined by the fraction of [32P]-label associated with the fragments representing dimerization relative to the total [32P]-label of each sample

as quantified after separation (for example, see Fig. 2). Experimental details are

described in the Methods Section for this DNA in the absence (■) and presence

of the histone octamer (△) and after reconstitution to form NCP (□).

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adjacent thymines to form the cyclobutane product (ESI†).

Finally, the photostationary state necessitates a product level

that can be achieved independently from any initial ratio of

starting material and product. This property was confirmed

using a DNA duplex of 47 base pairs that contained only a

single set of adjacent thymines. Within 40 min of irradiation,

cyclobutane thymine dimer yields remained a constant 5–10%

whether starting from the parent duplex or its analog containing 100% dimer (ESI†). Consequently, the levels of dimer

should reflect the composite variables affecting both its

formation and reversion under the conditions of these

experiments.

Neither the defined NCP generated with the 5S gene nor

the 601 sequence significantly affected dimer levels as illustrated in Fig. 3, 4 and 6. Each defined duplex provided an individual set of thymine pairs in specific locations within the

NCP. Such a narrow selection of DNA is a requirement for

maintaining consistent translational and rotational orientation

of the duplex, but this also severely restricts the variations

available for screening. The two sequences of this investigation

Fig. 4 Accumulation of individual cyclobutane thymine dimers (T^T) within the 5S rRNA gene (X. borealis). Fragmentation at each potential cyclobutane dimer site

in the absence (■) of the core histones and after reconstitution to form NCP (□) was monitored over time after endo V treatment and separation as illustrated in

Fig. 2. Yields are presented relative to total DNA. Individual measurements were averaged when determined repeatedly and error bars indicate the experimental

range of the data. Sites of dimerization are illustrated in Fig. 1.

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are negligible in number when compared to the 7 × 1019 possible sequences that were tested while surveying a randomized

region of 33 nucleotides for the effect of DNA looping on

thymine dimerization.25 Additionally, neither the 5S gene nor

the 601 sequence offered potential thymine dimer sites near

the dyad axis of NCP where distortion of the duplex is greatest

and dimerization is significantly suppressed in the heterogeneous array of sequences.23,24

Only the most reactive sequences of a mixed population are

characterized by product detection, and only a fraction of

those might respond to structural variation in DNA by a confluence of a particular nucleotide sequence and its associated

conformation, dynamics, excitation and relaxation. Access to

productive geometries appears key to efficient cyclobutane

dimerization as suggested by investigations using systems

ranging from simple model oligonucleotides to complex

protein DNA assemblies.22,25,57,58 Conversion of singlestranded to double-stranded DNA diminishes the available

range of possible nucleobase orientations as well as restricts

access to the most productive orientations for dimerization.18

Minor changes to the duplex structure then have the potential

to shift the population towards conformers with an enhanced

(or suppressed) ability to form cyclobutane thymine dimers as

evident from their periodic increase and decrease in duplex

DNA held in a loop.25

Neighboring sequences are known to modulate the

efficiency of thymine dimerization and reversion as well, and

the magnitude of their effect is dependent on structure or at

least requires duplex formation to exert its influence.41,42 Little

diversity is evident in the nucleotides neighboring the thymine

pairs within the 5S gene (Fig. 1). Most are surrounded by pyrimidines. The 601 sequence contains more thymine pairs surrounded by purines (Fig. 5), but still the local sequence in

these examples may only set their basal level of dimerization

and not necessarily respond to structural perturbations. This

is evident from the 5-fold difference in cyclobutane dimerization of thymines between complementary strands of a 175

base pair fragment of the 5S gene despite the similar number

of thymine pairs in each strand.50 Still, these differences

in reactivity were maintained whether the duplex was free in

solution or associated with the core histones.

The lack of NCP-dependent variation in dimer accumulation for the 5S gene and 601 sequence may also reflect a

minimal change in helix structure when free in solution versus

assembled into a NCP. This possibility is consistent with previous studies based on circular dichroism of the 601

sequence.59 These sequences avidly bind and orient along the

histone surface which implies a predisposition to adopt their

protein-bound conformation even in the absence of protein.

Even a pyrimidine tract inserted in the 5S gene at the dyad

axis, the region of greatest distortion, exhibited only minimal

change in thymine dimerization in the presence and absence

of the NCP.50 Perhaps the weaker binding of other sequences

to core histones indicates their need for a much greater distortion in conformational before accommodation into the NCP.

The magnitude of these perturbations may then modulate the

response of thymine dimerization to binding and looping

around proteins.

The structural perturbations generated by cyclobutane

thymine dimers had the additional potential to influence the

net accumulation of product under photostationary conditions

without affecting its initial rate of formation. The helical kink

generated by a thymine dimer is not expected to be equally tolerated in all rotational and translational settings of the NCP

and could therefore contribute to the periodic increase and

decrease of thymine dimers. However, the presence of a dimer

in the 5S gene only slightly reduced its affinity for histones

relative to that of the undamaged gene as published previously.60 Destabilization by the dimer was also not sufficient

to alter the orientation of this DNA with respect to the

histones.50,61 Only the rate of DNA release from a NCP based

on the 5S gene appeared to increase in the presence of the

Fig. 5 Translational and rotational orientation of the 601 DNA sequence

within the nucleosome core particle. Nucleobases are closest to the histone

octamer at the minima and farthest at the maxima of the line above the

DNA sequence. Sites of potential thymine dimerization are indicated as (1)

through (6).

Fig. 6 Time-dependent formation of the cyclobutane thymine dimer in the

601 DNA sequence. Total incorporation of dimers (T^T) was determined by the

fraction of [32P]-label associated with the fragments representing dimerization

relative to the total [32P]-label of each sample as quantified after separation (see

ESI, Fig. S1†). Experimental details are described in the Methods Section for this

DNA in the absence (■) of the histone octamer and after reconstitution to form

NCP (□). The lines are added only to highlight the trends of dimer formation.

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dimer.62 Thus, the insensitivity of the photostationary level of

cyclobutane thymine dimer to NCPs formed by the 5S gene

and 601 sequence is consistent with this lack of an observed

reorganization or distortion around the dimer. Identifying the

sequences that express the greatest range of photochemical

reactivity when incorporated into a NCP will require a future

strategy that combines the variable reactivity of heterogeneous

populations of DNA with the structural details of individual

constituents.

Acknowledgements

This research was supported in part by the National Science

Foundation (SER) a SMART Fellowship from the Department

of Defense (ASF), and a NSF CAREER Award (WBD). We thank

Prof. John-Stephen Taylor and Dr Matthew Ray Holman for

preparing and characterizing the duplex DNA containing a

single cyclobutane thymine dimer within a defined sequence.

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