8-oxoguanine lesioned B-DNA molecule complexed with repair enzyme hOGG1: a molecular dynamics study

The molecular dynamics (MD) simulation of DNA mutagenic oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), complexed with the repair enzyme, human oxoguanine glycosylase 1 (hOGG1), was performed for 1 nanosecond (ns) in order to describe the dynamical process of DNA-enzyme complex formation. After 900 picoseconds of MD the lesioned DNA and enzyme formed a complex that lasted until the end of the simulation at 1 ns. The complex was mainly represented by the overlapping van der Waals surfaces of DNA and enzyme molecules. The amino group of arginine 324 was located close to the phosphodiester bond of the nucleotide with 8-oxoG enabling chemical reactions between amino acid and lesion. The broken hydrogen bonds resulting in locally collapsed B-DNA structure were observed at the lesion site. The phosphodiester bond at C5' of 8-oxoG was displaced to the position close to the amino group of arginine 324. The water-mediated hydrogen bond network was formed in each contact area between DNA and enzyme, further enhancing the stability of the complex. In the background simulation of the identical molecular system with the native DNA, neither the complex nor the water- mediated hydrogen bond network was observed.     

Electrostatic energy analysis of 8-oxoguanine DNA lesion-molecular dynamics study

One nanosecond molecular dynamics (MD) simulation was performed for two DNA segments each composed of 30 base pairs. In one DNA segment the native guanines at nucleotides positions 17 and 19 were replaced with two 8-oxoguanines (8-oxoG) (8-oxoG is mutagenic DNA oxo-lesion). The analysis of results was focused on the electrostatic energy that is supposed to be significant factor causing the disruption of DNA base stacking in DNA duplex and may also serve as a signal toward the repair enzyme informing the presence of the lesion. The repulsive interaction between 8-oxoG and the entire DNA molecule was observed, which caused the extrahelical position of 8-oxoG (position 19). The repulsive electrostatic interaction between both 8-oxoG lesions contributed to the flipping out of one 8-oxoG and to the local instability of the lesioned DNA region. The electrostatic potential at the surface of DNA close to the lesions has more negative value than the same region on the native DNA. This electrostatic potential may signal presence of the lesion to the repair enzyme. In the simulation of native DNA segment, no significant structural changes were observed and B-DNA structure was well preserved throughout the MD simulation.     

Electrostatic energy analysis of 8-oxoguanine DNA lesion—molecular dynamics study

One nanosecond molecular dynamics (MD) simulation was performed for two DNA segments each composed of 30 base pairs. In one DNA segment the native guanines at nucleotides positions 17 and 19 were replaced with two 8-oxoguanines (8-oxoG) (8-oxoG is mutagenic DNA oxo-lesion). The analysis of results was focused on the electrostatic energy that is supposed to be significant factor causing the disruption of DNA base stacking in DNA duplex and may also serve as a signal toward the repair enzyme informing the presence of the lesion. The repulsive interaction between 8-oxoG and the entire DNA molecule was observed, which caused the extrahelical position of 8-oxoG (position 19). The repulsive electrostatic interaction between both 8-oxoG lesions contributed to the flipping out of one 8-oxoG and to the local instability of the lesioned DNA region. The electrostatic potential at the surface of DNA close to the lesions has more negative value than the same region on the native DNA. This electrostatic potential may signal presence of the lesion to the repair enzyme. In the simulation of native DNA segment, no significant structural changes were observed and B-DNA structure was well preserved throughout the MD simulation.     

The Influence of 5′,8-Cyclo-2′-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study

The 5′,8-cyclo-2′-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered—the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5′-end side of cdPu than on its 3′-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.     

Dynamic behavior of DNA base pairs containing 8-oxoguanine

The process by which DNA repair enzymes recognize and selectively excise damaged bases in duplex DNA is fundamental to our mechanistic understanding of these critical biological reactions. 8-Oxoguanine (8-oxoG) is the most common form of oxidative DNA damage; unrepaired, this lesion generates a G:C-->T:A mutation. Central to the recognition and repair of DNA damage is base extrusion, a process in which the damaged base lesion or, in some cases, its partner disengages from the helix and is bound to the enzyme's active site where base excision takes place. The conformation adopted by 8-oxoG in duplex DNA is affected by the base positioned opposite this lesion; conformational changes may also take place when the damaged base binds to its cognate repair enzyme. We performed unrestrained molecular dynamics simulations for several 13-mer DNA duplexes. Oligomers containing G:C and 8oxoG:C pairs adopted Watson-Crick geometries in stable B-form duplexes; 8oxoG showed increased local and global flexibility and a reduced barrier to base extrusion. Duplexes containing the G:A mismatch showed much larger structural fluctuations and failed to adopt a well-defined structure. For the 8oxoG:A mismatch that is recognized by the DNA glycosylase MutY, the damaged nucleoside underwent spontaneous and reproducible anti-->syn transitions. The syn conformation is thermodynamically preferred. Steric hindrance and unfavorable electrostatics associated with the 8oxoG O8 atom in the anti conformation were the major driving forces for this transition. Transition events follow two qualitatively different pathways. The overall anti-->syn transition rate and relative probability of the two transition paths were dependent on local sequence context. These simulations indicate that both the dynamic and equilibrium behavior of the duplex change as a result of oxidation; these differences may provide valuable new insight into the selective action of enzymes on damaged DNA.     

Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC–MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.     

8-Oxoguanine enhances bending of DNA that favors binding to glycosylases

Molecular dynamics (MD) simulations were carried out on the DNA oligonucleotide GGGAACAACTAG:CTAGTTGTTCCC in its native form and with guanine in the central G(19):C(6) base pair replaced by 8-oxoguanine (8oxoG). A box of explicit water molecules was used for solvation, and Na(+) counterions were added to neutralize the system. The direction and magnitude of global bending were assessed by a technique used previously to analyze simulations of DNA containing a thymine dimer. The presence of 8oxoG did not greatly affect the magnitude of DNA bending; however, bending into the major groove was significantly more probable when 8oxoG replaced G(19). Crystal structures of glycosylases bound to damaged-DNA substrates consistently show a sharp bend into the major groove at the damage site. We conclude that changes in bending dynamics that assist the formation of this kink are a part of the mechanism by which glycosylases of the base excision repair pathway recognize the presence of 8oxoG in DNA.     

Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA

The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.     

Molecular dynamics simulation of thymine glycol-lesioned DNA reveals specific hydration at the lesion

One nanosecond molecular dynamics (MD) simulation of a thymine glycol (TG)-lesioned part of human lymphoblast AG9387 was performed to determine structural changes in DNA molecule caused by the presence of a lesion. These changes can be significant for proper recognition of lesions by a repair enzyme. Thymine glycol is the DNA oxidative lesion formed by addition of OH radicals to C5 and C6 atoms of the thymine base. This lesion is known as causing Cockayne Syndrome-inherited genetic disorder. Distribution of water molecules in a hydration shell around the DNA molecule was analyzed for its contribution to the recognition of the TG lesion by the repair enzyme. The results of MD simulation show there is a specific DNA structural configuration formed at the lesion. After 500 ps the DNA is bent in a kink at the TG site. This change dislocates the glycosyl bond at C5' to a position closer to the DNA surface, and thus its atoms are more exposed to the surrounding water shell. The increased number of water molecules that are close to the TG site indicates that the glycosyl bond may be easily contacted by the repair enzyme. In addition, the higher number of water molecules at the TG site substantiates the importance of water-mediated hydrogen bonds created between the repair enzyme and the DNA upon formation of the complex. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1723-1731, 2001     

When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.     

In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as “clustered/complex DNA damage” and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.     

Damage control of DNA in nucleosome core particles: when a histone's loving, protective embrace is just not good enough

Packaging DNA into nucleosome core particles generally offers protection from damage by molecules diffusing in solution. However, on page 403 of this issue, Barton and coworkers report that although noncovalently bound, activated Rh (Rhodium) does not readily bind within nucleosomal DNA, activated Rh that is covalently tethered to the 5' terminus of a histone-associated oligonucleotide oxidizes guanine bases from a distance of up to 24 base pairs, demonstrating that histones do not protect DNA from long-range damage from the transport of charge through stacked bases. This implies that oxidative damage generated on DNA in vivo may spread from an initially damaged site to distal sites. Once created, such sites may persist and be resistant to repair because of the protective packaging by histones; they thus may result in permanent mutations.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

8-oxoguanine and 8-oxodeoxyguanosine Biomarkers of Oxidative DNA Damage: A Review on HPLC–ECD Determination

Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2′-deoxyguanosine (8-oxodG)—the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC–ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC–ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.     

基于分子信标对hOGG1活性的定量分析

设计了一种含有8-oxoG碱基的新型分子信标, 结合酶促反应发展了一种非同位素标记的人8-oxoG-鸟嘌呤糖苷酶1(hOGG1)的活性分析新方法, 检出限可达0.0125 U/mL. 此外, 该方法还可用于快速考察金属离子对酶促反应的影响和肿瘤细胞中hOGG1活性水平的定量检测. 实验结果表明, 该方法简单、 灵敏, 有望用于肿瘤样品中hOGG1活性的高通量分析和hOGG1抑制剂的筛选.     

Possible cause of G-C-->C-G transversion mutation by guanine oxidation product, imidazolone

BACKGROUND: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G-C-->T-A and G-C-->C-G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G-C-->T-A transversion mutations. Although the number of G-C-->C-G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G-C-->C-G transversions is not known. RESULTS: To determine which oxidative products are responsible for G-C-->C-G transversion mutations, we photooxidized 5'-d(AAAAAAGGAAAAAA)/5'-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4 degrees C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2'-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4H-imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz-G base pairs are formed. The 'reverse' approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz-G base pair. CONCLUSIONS: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz-G base pair formation can explain the G-C-->C-G transversion mutations that appear under oxidative conditions.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

PostExcision Events in Human Nucleotide Excision Repair

The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage‐containing DNA oligonucleotide and a single‐stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single‐stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less‐explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.     

Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro-tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA pho-toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro-sophila, Xenopus and Crotalus.