Cis-syn Cyclobutane Pyrimidine Dimer Research Articles

ИНТЕРПРЕТАЦИЯ ГОРЯЧИХ ПЯТЕН УЛЬТРАФОЛЕТОВОГО МУТАГЕНЕЗА, ОБРАЗУЮЩИХСЯ НА ЗАПАЗДЫАЮЩЕЙ НИТИ ДВУХЦЕПОЧЕЧНОЙ ДНК ГЕНА supF

At present, the mechanism of formation of hot and cold spots of ultraviolet mutagenesis is not clear. I developed a polymerase-tautomeric model of the mechanism of formation of hot and cold spots of ultraviolet mutagenesis and showed that the probability of mutation formation depends on the processes of propagation of excitation energy along the DNA molecule. In my proposed polymerase-tautomeric model of ultraviolet mutagenesis, it was shown that mutations are formed opposite only those cis-syn cyclobutane pyrimidine dimers, one or both of which are in rare tautomeric forms. In the polymerase-tautomeric model of the mechanism of formation of hot and cold spots of ultraviolet mutagenesis, I have shown that the hot spots of ultraviolet mutagenesis are those cis-syn cyclobutane pyrimidine dimers to which the most excitation energy is transferred. In a number of works, I calculated the relative probabilities of mutations formed opposite the DNA bases that are part of the cis-syn cyclobutane pyrimidine dimers that appeared upon irradiation of double-stranded DNA of the supF gene. In this article, based on the results of previous calculations, I interpret experimental data in which hot spots of ultraviolet mutagenesis are DNA regions consisting of three or more pyrimidine DNA bases arranged in a row.

Genome-wide profiles of UV lesion susceptibility, repair, and mutagenic potential in melanoma.

Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54( (34), ), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

Global Deformation of DNA Facilitates Recognition and Nucleotide Flipping of Damaged Sites: A Molecular Dynamics Simulation Study

Nucleotide excision repair of damaged DNA requires recognition of the DNA lesion and a conformational transition of damaged nucleotides from an intra-helical to an extra-helical conformation. The extra-helical damaged nucleotides can enter the active site of a repair enzyme. For the frequent cis-syn-cyclobutane pyrimidine dimer (CPD) damage we performed extensive molecular dynamics (MD) simulations indicating that it adopts transient conformations which resemble the global shape of the repair enzyme bound conformation more closely compared to regular B-DNA. It is likely that this transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out (flipping) process of the damaged DNA. Furthermore, umbrella sampling free energy simulations to loop out the damaged site indicate that the global DNA deformation observed in the enzyme bound form facilitates the flipping process (lowers energy penalty) even in the absence of the repair enzyme. Similar results were found for the flipping process of oxidized Guanine (8-oxo-Guanine) another common DNA damage. The result indicates that the DNA deformation induced by the repair enzyme binding has a significant influence on the flipping process. The additional effect of protein-DNA contacts on the calculated free energy for the flipping process will also be presented.

Translesion synthesis past guanine(C8)-thymine(N3) intrastrand cross-links catalyzed by selected A- and Y-family polymerases.

Oxidatively generated guanine radicals in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. These G[8-3]T lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defence mechanisms. The abilities of several representative polymerases to bypass the G[8-3]T lesions in two different sequence contexts, G*T* and G*CT*, were assessed in vitro. The polymerase BF (bacillus fragment) from Bacillus stearothermophilus, the Y-family archaeal polymerases Dpo4 from Sulfolobus sulfataricus P2, and human DNA pol κ and pol η were selected for the study. The A-family polymerase BF was strongly blocked, while relatively weak translesion synthesis was observed in the case of Y-family polymerases Dpo4 and pol κ. Primer extension catalyzed by pol η was also partially stalled at various positions at or near the G[8-3]T cross-linked bases, but a significant and distributive primer extension was observed beyond the sites of the lesions with the efficiency being consistently greater in the case of G*CT* than in the case of G*T* lesions. The results obtained with pol η are compared with translesion synthesis past other intrastrand cross-linked lesions with previously published results of others that include the isomeric G[8-5m]T lesions generated by ionizing radiation, the cis-syn cyclobutane pyrimidine dimer and the 6-4 photoproduct generated by UV irradiation, and the Pt-G*G* lesions derived from the reactions of the chemotherapeutic agent cisplatin with DNA.

Recognition of Damaged DNA for Nucleotide Excision Repair: A Correlated Motion Mechanism with a Mismatched cis-syn Thymine Dimer Lesion.

Mammalian global genomic nucleotide excision repair requires lesion recognition by XPC, whose detailed binding mechanism remains to be elucidated. Here we have delineated the dynamic molecular pathway and energetics of lesion-specific and productive binding by the Rad4/yeast XPC lesion recognition factor, as it forms the open complex [Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570–575; Chen, X., et al. (2015) Nat. Commun. 6, 5849] that is required for excision. We investigated extensively a cis-syn cyclobutane pyrimidine dimer in mismatched duplex DNA, using high-level computational approaches. Our results delineate a preferred correlated motion mechanism, which provides for the first time an atomistic description of the sequence of events as Rad4 productively binds to the damaged DNA.

Influence of a cis,syn-cyclobutane pyrimidine dimer damage on DNA conformation studied by molecular dynamics simulations.

The photo-induced formation of cis-syn-cyclobutane pyrimidine dimers (CPD) is a highly mutagenic and cancerogenic DNA lesion. In bacteria photolyases can efficiently reverse the dimer formation employing a light-driven reaction after looping out the CPD damaged bases into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The CPD damage may alter the DNA structure and dynamics already in the absence of the repair enzyme which can facilitate the initial binding of a photolyase repair enzyme. To characterize the effect of a CPD damage, extensive comparative molecular dynamics (MD) simulations on duplex DNA with central regular or CPD damaged nucleotides were performed supplemented with simulations of the DNA-photolyase complex. Although no spontaneous flipping out transitions of the damaged bases were observed, the simulations showed significant differences in the conformational states of regular and CPD damage DNA. The isolated damaged DNA adopted transient conformations which resembled the global shape of the repair enzyme bound conformation more closely compared to regular B-DNA. In particular, these conformational changes were observed in most of helical and structural parameters where the protein bound DNA differs drastically from regular B-DNA. It is likely that the transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out process of the damaged DNA.

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells.

DNA polymerase eta (Pol η) is one of several Y family trans-lesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased both with empty vector control (EVC), wild-type POLH, and Y52E-POLH over 48 hours post UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV induced DNA lesions in HaCaT cells.

Biochemical analysis of active site mutations of human polymerase η

DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis–syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called ‘damage tolerance’ to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine–thymine CPD (T–T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T–T CPD.

DNA photolyases and SP lyase: structure and mechanism of light-dependent and independent DNA lyases

Light is essential for many critical biological processes including vision, circadian rhythms, photosynthesis and DNA repair. DNA photolyases use light energy and a fully reduced flavin cofactor to repair the major UV-induced DNA damages, the cis-syn cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproducts. Catalysis involves two photoreactions, the photoactivation which leads to the conversion of the flavin cofactor to its catalytic active form and the photorepair whose efficiency depends on a light-harvesting antenna chromophore. Very interestingly, an alternative and light-independent direct reversal mechanism to repair a distinct photolesion is found in bacterial spores, catalyzed by spore photoproduct lyase. This radical SAM enzyme uses an iron-sulfur cluster and S-adenosyl-l-methionine (SAM) to split a specific photoproduct, the so-called spore photoproduct (SP), back to two thymidine residues. The recently solved crystal structure of SP lyase provides new insights into this unique DNA repair mechanism and allows a detailed comparison with DNA photolyases. Similarities as well as divergences between DNA photolyases and SP lyase are highlighted in this review.

Effects of DNA lesions on the transcription reaction of mitochondrial RNA polymerase: implications for bypass RNA synthesis on oxidative DNA lesions

Oxidative DNA lesions inhibit the transcription of RNA polymerase II, but in the presence of transcription elongation factors, the transcription can bypass the lesions. Single-subunit mitochondrial RNA polymerase (mtRNAP) catalyses the synthesis of essential transcripts in mitochondria where reactive oxidative species (ROS) are generated as by-products. The occurrence of RNA synthesis by mtRNAP at oxidative DNA lesions remains unknown. Purified mtRNAP and a complex of RNA primer/DNA template containing a single DNA lesion, such as ROS-induced 8-oxoguanine (8-oxoG), two isomeric thymine glycols (5R-Tg or 5S-Tg), the UV-induced cis-syn cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone photoproduct (6-4pp), or a spontaneous common DNA lesion, a base-loss-induced apurinic/apyrimidinic (AP) site, were used for in vitro RNA synthesis assays. In this report, we show that mtRNAP bypassed the oxidative DNA lesions of non-bulky 8-oxoG and 5R-Tg and 5S-Tg with pausing sites but did not bypass the UV-induced DNA lesions and the AP site. The bacteriophage T7 phage RNA polymerase, which is homologous to mtRNAP, bypassed 8-oxoG but stalled at 5R-Tg and 5S-Tg. As expected, although translesion RNA synthesis in 8-oxoG on the DNA templates generated incorrect transcripts with a G:C to T:A transversion, the synthesis in Tg could lead to the correct transcripts with no transcriptional mutagenesis. Collectively, these data suggest that mtRNAP may tolerate the mitochondrial genome containing oxidative DNA lesions induced by ROS from the side effects of an ATP generation reaction.

Evidence from Thermodynamics that DNA Photolyase Recognizes a Solvent-Exposed CPD Lesion

Binding of a cis,syn-cyclobutane pyrimidine dimer (CPD) to Escherichia coli DNA photolyase was examined as a function of temperature, enzyme oxidation state, salt, and substrate conformation using isothermal titration calorimetry. While the overall ΔG° of binding was relatively insensitive to most of the conditions examined, the enthalpic and entropic terms that make up the free energy of binding are sensitive to the conditions of the experiment. Substrate binding to DNA photolyase is generally driven by a negative change in enthalpy. Electrostatic interactions and protonation are affected by the oxidation state of the required FAD cofactor and substrate conformation. The fully reduced enzyme appears to bind approximately two additional water molecules as part of substrate binding. More significantly, the experimental change in heat capacity strongly suggests that the CPD lesion must be flipped out of the intrahelical base stacking prior to binding to the protein; the DNA repair enzyme appears to recognize a solvent-exposed CPD as part of its damage recognition mechanism.

Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase

The repair of cis–syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge–charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure–function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.

Lesion bypass by S. cerevisiae Pol ζ alone

DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

Role of Human DNA Polymerase κ in Extension Opposite from a cis–syn Thymine Dimer

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.

Characterization of a Y‐Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana

Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents.

Zebrafish have a competent p53-dependent nucleotide excision repair pathway to resolve ultraviolet B-induced DNA damage in the skin.

Ultraviolet (UV) light is a primary environmental risk factor for melanoma, a deadly form of skin cancer derived from the pigmented cells called melanocytes. UVB irradiation causes DNA damage, mainly in the form of pyrimidine dimers (cis-syn cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts), and organisms have developed complex multiprotein repair processes to cope with the DNA damage. Zebrafish is becoming an important model system to study the effects of UV light in animals, in part because the embryos are easily treated with UV irradiation, and the DNA damage repair pathways appear to be conserved in zebrafish and mammals. We are interested in exploring the effects of UV irradiation in young adult zebrafish, so that we can apply them to the study of gene-environment interactions in models of skin cancer. Using the Xiphophorus UV melanoma model as a starting point, we have developed a UV irradiation treatment chamber, and established UV treatment conditions at different ages of development. By translating the Xiphophorus UV treatment methodology to the zebrafish system, we show that the adult zebrafish skin is competent for nucleotide excision DNA damage repair, and that like in mammalian cells, UV treatment promotes phosphorylation of H2AX and a p53-dependent response. These studies provide the groundwork for exploring the role of UV light in melanoma development in zebrafish.

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV-light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis-syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6-4)pyrimidone photoproducts ((6-4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6-4) lesions into strained four-membered ring repair intermediates, it is surprising that the not rearranged T(6-4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6-4) DNA photolyases.