The DNA damage spectrum produced by simulated sunlight

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.     

Rapid repair kinetics of pyrimidine(6-4)pyrimidone photoproducts in human cells are due to excision rather than conformational change.

UV-induced pyrimidine(6-4)pyrimidone photoproducts in DNA of mammalian cells are apparently repaired much more rapidly than cyclobutane dimers. Since only immunological assays for (6-4) photoproducts have been sensitive enough for repair measurements, it was possible that these apparently rapid repair kinetics reflected a change in physical conformation of antibody-binding sites, resulting in epitope loss rather than excision. To discriminate between these possibilities, we developed a procedure to photochemically convert (6-4) photoproducts to single-strand breaks in UV-irradiated DNA with a background low enough to permit repair measurements. Analysis of a specific DNA sequence indicated that photoinduced alkali-labile sites (PALS) were induced with the same site-specificity as (6-4) photoproducts. Normal human and xeroderma pigmentosum (XP) variant cells rapidly excised (6-4) photoproducts measured as PALS, but little repair was seen in cells from XP complementation group A. These repair kinetics corresponded to those determined in the same samples by radioimmunoassay of (6-4) photoproducts. Thus we conclude that the rapid repair of (6-4) photoproducts observed in UV-irradiated human cells is not the result of a conformational change resulting in epitope loss, but reflects excision of this photoproduct from DNA.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light.

Ultraviolet (UV) light induces a variety of lesions in DNA of which the pyrimidine dimer represents the major species. Pyrimidine dimers exist as both a cyclobutane type and a 6-4' (pyrimidine-2'-one) photoproduct. We have purified a protein of M(r) approximately 125,000 from HeLa cell nuclei which binds efficiently to double-stranded DNA irradiated with UV light but not to undamaged DNA. This protein was designated UVBP1 (UV damage binding protein 1). UVBP1 did not recognise DNA damaged by cisplatin. Using oligonucleotides with a single dipyrimidine site for induction of UV photoproducts, binding of UVBP1 to a TC-containing substrate was shown to be more efficient than to substrates containing a TT, a CT or a CC pair. This binding specificity implies selective recognition of the 6-4' photoproduct. Further evidence for this was provided by the finding that hot alkali treatment of the substrate (which selectively hydrolyses 6-4' photoproducts) abrogated binding of UVBP1, whereas incubation with DNA photolyase to remove cyclobutane dimers did not. No detectable DNA helicase, ATPase or exonuclease activity was associated with the purified protein. We suggest that UVBP1 may be involved in the lesion recognition step of DNA excision repair and could contribute to the preferential repair of 6-4' photoproducts from the DNA of UV-irradiated mammalian cells.     

Base sequence specificity of a monoclonal antibody binding to (6-4)photoproducts.

The DNA base sequence specificity of the 64M-1 monoclonal antibody, which recognizes ultraviolet (UV)-induced (6-4)photoproducts, was characterized. The 64M-1 antibody strongly bound to UV-poly(dU) as well as to UV-poly(dT), and weakly to UV-poly(dC), UV-poly(me5dC) and UV-poly(rU). A competitive inhibition assay using UV-oligo(dT)8, UV-oligo(dTdC)4, UV-oligo(dC)8, UV-PvuI linker (GCGATCGC) and UV-PvuII linker (GCAGCTGC) indicated that the main (6-4)photoproducts detected by the 64M-1 antibody in UV-irradiated DNA are TT(6-4)photoproducts and TC(6-4)photoproducts. Comparison between dTpdT(6-4)photoproduct and dTpdC(6-4)photoproduct showed that the affinity of the 64M-1 antibody for dTpdT(6-4)photoproduct was about 5 times higher than that for dTpdC(6-4)photoproduct. The antibody also binds to isolated TT(6-4)photoproducts.     

Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.

Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).     

The induction and repair of lesions produced by the photolysis of (6-4) photoproducts in normal and UV-hypersensitive human cells

A radioimmunoassay was used to study the induction and repair of damage produced by the photolysis of (6-4) photoproducts in normal and UV-sensitive human cells. Photochemical conditions were established to optimize the production of photolyzed (6-4) photoproducts in human cell DNA with minimal induction of other photoproducts. The repair of this photoproduct, presumed to be a Dewar pyrimidinone, was similar to that determined for the (6-4) photoproduct, with most of the antibody-binding sites removed within 4 h post-photolysis. Whereas xeroderma pigmentosum group A cells were deficient in the repair of this lesion, an XP variant and two cell lines selectively hypersensitive to UVB-irradiation were shown to have normal repair. The radioimmunoassay was further used to demonstrate the alkali-lability of the (6-4) photolysis product.     

A comparison of UV induced DNA photoproducts from isolated and non-isolated developing bacterial forespores

UV-induced photoproduct formation has been compared in non-isolated and isolated developing forespores. We have found that levels of spore type photoproducts are greatly affected by mother cell DNA. We have also observed the presence of the photoproduct 6-4′-(pyrimidin-2′-one)-thymine in developing forespores. We conclude from these and other data in the literature that the degree of hydration around the forespore DNA is reduced by the presence of dipicolinic acid which influences photoproduct formation without causing a change in conformational state.     

UV-induced photoproducts of 5-methylcytosine in a DNA sequence context.

In order to detect possible m5C photoproducts, highly purified rat liver DNA-cytosine methyltransferase was used to specifically generate m5C with a radioactive methyl group. When these DNAs were subjected to a large dose (10 kJ/m2) of 254 nm or 302 nm ultraviolet light (UVB) to enhance the yield, two labeled photoproducts were detected and isolated by reverse phase HPLC after formic acid hydrolysis. Further studies using acetone as a triplet state sensitizer and UVB irradiation suggested that photoproduct II was activated via a triplet state while the more polar photoproduct I was not. Photoreversion of the purified photoproducts with 10 kJ/m2 254 nm light demonstrated the following reactions: Photoproduct I regenerated m5C, while photoproduct II is split and regenerated m5C and photoproduct I. These results suggest that photoproduct I is monomeric while photoproduct II dimeric, and from the latter's elution position possibly a cyclobutyl type dimer arising from a reaction with an adjacent cytosine. Using d[TTG] and d[Cm5CG] as models of typical sequences, irradiation with 10 kJ/m2 254 nm or 302 nm, respectively, gave rise to a small component having altered mobility in sequencing gels. The altered mobility trinucleotides were resistant to degradation by PI and micrococcal nucleases as expected from photodimerization of the pyrimidine bases. Furthermore, oligonucleotide substrates containing m5C were synthesized and shown to be susceptible to T4 endonuclease v action at locations consistent with d[Cm5C] photodimer formation when irradiated in the UVB range.     

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

Use of a combined enzymatic digestion/ESI mass spectrometry assay to study the effect of TATA-binding protein on photoproduct formation in a TATA box

Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproduct formation in a TATA box under UVC irradiation [Aboussekhra and Thoma (1999) EMBO J. 18, 433-443]. The conclusions of that study were based on an indirect enzymatic assay that was not specific for (6-4) photoproducts. Herein we report the use of a recently developed coupled enzymatic digestion/mass spectrometry assay [Wang et al. (1999) Chem. Res. Toxicol. 12, 1077-1082] to identify unambiguously and quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in the presence or absence of TBP binding. Exposure of the adenovirus major late promoter TATA box to a high dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominant formation of the cis-syn dimer within the T(4) tract, whereas exposure in the presence of TBP leads to almost exclusive formation of the (6-4) photoproduct. In contrast, the (6-4) product is not detected at high doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although the cis-syn product predominates. When the products of UVB irradiation were subsequently exposed to a high dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly the exclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light. Both cis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with small doses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose. Through the use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4) photoproducts formed most selectively at the second and third positions of the T(4) tract upon either UVB or UVC irradiation in the presence of TBP. By using the same substrate, it was found that UVC-induced TA formation was inhibited by TBP binding and that TA formation was greatest at the 5' end of the TATA sequence.     

The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli

Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV-induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV-induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light-induced mutations such as deletions. None of the available evidence rules out (6-4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS)     

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.     

Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts

We obtained a monoclonal antibody directed against UV-induced DNA damage. Analysis of the antigenic determinant in UV-irradiated DNA recognized by this antibody, 64M-1, revealed that it bound UV-irradiated oligo- or poly-nucleotides containing thymine-thymine or thymine-cytosine sequences. The antibody failed to bind DNA irradiated with 313 nm UV in the presence of acetophenone, which contained predominantly thymine dimers as DNA damage. The binding activity of this antibody to 254-nm UV-irradiated DNA decreased with 313-nm UV irradiation, and the decrease of this binding activity correlated with the decrease of fluorescence corresponding to (6-4) photoproducts. These results suggest that the antigenic determinant recognized by this monoclonal antibody is a (6-4) photoproduct. Using autoradiography with 3H-antibody, we could detect the formation of the (6-4) photoproduct in individual human cells irradiated with 254-nm UV doses as low as 20 J/m2.     

An ultraviolet light-damaged DNA recognition protein absent in xeroderma pigmentosum group E cells binds selectively to pyrimidine (6-4) pyrimidone photoproducts.

The binding specificity was defined of a human ultraviolet light-damaged DNA recognition protein (UV-DRP), the activity of which is absent in some xeroderma pigmentosum complementation group E cells. Our results suggest that cyclobutane pyrimidine dimers (CPDs) are not high affinity UV-DRP binding sites--a finding consistent with other reports on this protein (Hirschfeld et al., (1990) Mol. Cell Biol., 10, 2041-2048). A major role for 6-4 photoproducts in UV-DRP binding was suggested in studies showing that irradiated oligonucleotides containing a T4C UV box sequence, which efficiently forms a TC 6-4 photoproduct, was a superior substrate for the UV-DRP when compared to a similar irradiated oligonucleotide having a T5 sequence. The latter sequence forms CPDs at a much higher frequency than 6-4 photoproducts. In a more direct approach, T4C-containing oligonucleotides complexed with the UV-DRP were separated from the unbound oligonucleotide fraction and the frequencies of 6-4 photoproducts in the two DNA populations were compared. The UV-DRP-bound fraction was highly enriched for the 6-4 lesion over the unbound fraction supporting the conclusion that 6-4 photoproducts are the principal binding cues for the UV-DRP.