Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics

Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site. In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X-ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.     

AP site structural determinants for Fpg specific recognition.

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.     

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.     

Low levels of endogenous oxidative damage cluster levels in unirradiated viral and human DNAs

Ionizing radiation induces bistranded DNA damage clusters-two or more oxidized bases, abasic, sites or strand breaks on opposing strands within a few helical turns-but it is not known if clusters are also formed in unirradiated DNA in solution or in unirradiated cultured human cells. The frequencies of endogenous oxidized purine clusters (recognized by Escherichia coli Fpg protein), oxidized pyrimidine clusters (recognized by Nth protein), and abasic clusters (cleavage by Nfo protein) were determined using quantitative gel electrophoresis, electronic imaging, and number average length analysis. Methods of DNA isolation and storage were found to affect cluster levels significantly. In bacteriophage T7 DNA prepared using stringent conditions, the frequencies of these clusters were <1/Mbp. In DNA from unirradiated human 28SC monocytes, the levels of such clusters were, at most, a few per gigabase pair.     

Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.     

Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli.

Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups. In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites. Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases. The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination. The release of 5'-terminal dRp by crude extracts of E. coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.     

Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages.     

Response of a DNA-binding protein to radiation-induced oxidative stress

The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis.Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins.When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently.The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.     

Computational analysis of the mode of binding of 8-oxoguanine to formamidopyrimidine-DNA glycosylase

8-Oxoguanine (8OG) is the most prevalent form of oxidative DNA damage. In bacteria, 8OG is excised by formamidopyrimidine glycosylase (Fpg) as the initial step in base excision repair. To efficiently excise this lesion, Fpg must discriminate between 8OG and an excess of guanine in duplex DNA. In this study, we explore the structural basis underlying this high degree of selectivity. Two structures have been reported in which Fpg is bound to DNA, differing with respect to the position of the lesion in the active site, one structure showing 8OG bound in the syn conformation and the other in the anti conformation. Remarkably, the results of our all-atom simulations are consistent with both structures. The syn conformation observed in the crystallographic structure of Fpg obtained from Bacillus stearothermophilus is stabilized through interaction with E77, a nonconserved residue. Replacement of E77 with Ser, creating the Fpg sequence found in Escherichia coli and other bacteria, results in preferred binding of 8OG in the anti conformation. Our calculations provide novel insights into the roles of active site residues in binding and recognition of 8OG by Fpg.     

Recognition and kinetics for excision of a base lesion within clustered DNA damage by the Escherichia coli proteins Fpg and Nth

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damages that are thought to lead to deleterious biological consequences, due to the challenge that clustered damage may present to the repair machinery of the cell. Specific oligonucleotides, containing either dihydrothymine (DHT) or 7,8-dihydro-8-oxoguanine (8-oxoG) opposite to specific lesions at defined positions on the complementary strand, have been used to determine the kinetic constants, K(M), k(cat), and specificity constants, for excision of DHT and 8-oxoG by the Escherichia coli base excision repair proteins, endonuclease III (Nth) and formamidopyrimidine glycosylase (Fpg), respectively. For excision of DHT opposite to 8-oxoadenine by Nth or Fpg proteins, or 8-oxoG opposite to 8-oxoG by Fpg, the major change in the specificity constant occurs when the second lesion on the complementary strand is one base to the site opposite to DHT or 8-oxoG. The specificity constants for excision of DHT or 8-oxoG by both proteins are reduced by up to 2 orders of magnitude when an abasic site or a strand break is opposite on the complementary strand. Whereas the values of K(M) are only slightly affected by the presence of a second lesion, the major change is seen as a reduction in the values of k(cat). The binding of Fpg protein to oligonucleotides containing 8-oxoG is inhibited, particularly when a single strand break is near to 8-oxoG on the complementary strand. It is inferred that not only the binding affinity of Fpg protein to the base lesion but also the rate of excision of the damaged base is reduced by the presence of another lesion, particularly a single strand break or an AP site on the complementary strand.     

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.     

Thermodynamics of the multi-stage DNA lesion recognition and repair by formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence--stopped-flow pre-steady-state kinetics

Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10-30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van't Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.     

114 Kinetic and thermodynamic basis for damaged bases excision by 8-oxoguanine-DNA glycosylases

DNA glycosylases play the opening act in a highly conserved process for excision of damaged bases from DNA called the base excision repair pathway. DNA glycosylases attend to a wide variety of lesions arising from both endogenous and exogenous factors. The types of damage include alkylation, oxidation, and hydrolysis. A major DNA oxidation product is 8-oxoguanine (8-oxoG), a base with a high mutagenic potential. In bacteria, this lesion is repaired by formamidopyrimidine-DNA glycosylase (Fpg), while in the case of humans this function belongs to 8-oxoG-DNA glycosylase (OGG1). We have attempted a comprehensive characterization of 8-oxoG recognition by DNA glycosylases. First, we have obtained thermodynamic parameters for melting of DNA duplexes containing 8-oxoG in all possible nucleotide contexts. The energy of stacking interactions of 8-oxoG was in strict dependence on 8-oxoG nucleotide environment, which may affect the recognition of damage and the efficiency of eversion of 8-oxoG from DNA helix by glycosylases. Next, we established how the flexibility of DNA context affects damage recognition by these enzymes (Kirpota et al., 2011). Then, we have found that DNA containing 8-oxoG next to a single-strand break provides a good substrate for Fpg, as soon as all structural phosphate residues are maintained. Using site-directed mutagenesis, we have addressed the functions of many previously unstudied amino acid residuess that were predicted to be important for Fpg activity by molecular dynamics simulation and phylogenetic analysis. Of note, many substitutions abolished the excision of 8-oxoG, but did not affect the cleavage efficiency of abasic substrates. Finally, we investigated the contribution of separated structural domains of Fpg to specific enzyme-substrate interaction. Surprisingly, despite the absence of the catalytic domain, C-terminal domain of Fpg possessed a low- residual ability to recognize and cleave abasic substrates. Our study sheds light on mechanism details of Fpg and OGG1 activity, with the ultimate goal of understanding how binding energy can be spent by these enzymes for catalysis.     

Stopped-flow kinetic studies of the interaction between Escherichia coli Fpg protein and DNA substrates

Formamidopyrimidine-DNA-glycosylase of Escherichia coli (Fpg protein) repairs oxidative DNA damage by removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. This enzyme possesses three types of activities resulting in the excision of oxidized residue from DNA: hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP-lyase), and delta-elimination. In our work, the kinetic mechanism for 8-oxoguanine excision from DNA substrate with Fpg protein has been determined from stopped-flow measurements of changes in the tryptophan fluorescence. The 12-nucleotide duplex d(CTCTC(oxo)GCCTTCC)*d(GGAAGGCGAGAG) containing the 8-oxoG nucleotide in the sixth position of one strand was used as the specific substrate. Four distinct phases in the time traces were detected. These four-phase transition changes in the Fpg protein fluorescence curves were analyzed by global fitting to determine the intrinsic rate constants. We propose that the first two phases represent the equilibrium steps. The first of them describes the bimolecular binding step and the second, formation of the apurinic site. The third, irreversible step is believed to describe the beta-elimination process. The fourth step reflects the delta-elimination and decomposition of complex between enzyme and the product of 8-oxoG nucleotide excision. The results obtained provide direct evidence of conformational transitions of the Fpg protein during the catalytic process. The significance of these results for the functioning of Fpg protein is discussed.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Radiation-induced binding of DNA from irradiated mammalian cells to hydroxyapatite columns

In experiments designed to measure radiation-induced DNA damage using the DNA unwinding-hydroxyapatite chromatography technique, we observed that under some experimental conditions a significant proportion of the test DNA became tightly bound to the hydroxyapatite (HA) and could not be released even with a high concentration of phosphate buffer. Approximately 5-10% of DNA from unirradiated cells binds to the HA. With increasing radiation doses in air, the fraction of bound DNA increases, reaching about 30% at about 35 Gy. The binding exhibits many of the characteristics of a radiation-induced cell lesion: the proportion of DNA retained by the HA is less when cells are irradiated under hypoxic conditions or in the presence of the thiol radioprotector dithiothreitol; and the binding decreases when an incubation period is allowed between irradiation and harvest of the cells for assay. Studies to determine the nature of the lesion responsible for the binding demonstrated that lesion production requires a component found in cells since no binding was observed with irradiated isolated DNA or nuclear matrix; the binding is not a result of the production of DNA-protein crosslinks; and the bound DNA is single-stranded, based on its sensitivity to nuclease S1. Because of the dose dependence of the binding of DNA to HA, the slopes of the dose-response curves for DNA damage determined with this assay depend on the method used to calculate the fraction of double-stranded DNA. Our demonstration that the bound DNA is single-stranded guides the choice of the method for data analysis.     

Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein

The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity.     

Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA

The formamidopyrimidine-DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.     

Structure and Dynamics of DNA Duplexes Containing a Cluster of Mutagenic 8-Oxoguanine and Abasic Site Lesions

Clustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used ¹H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3′ or the 5′ side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process.     

Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening

Telomere shortening is associated with cellular senescence. We investigated whether UVA, which contributes to photoaging, accelerates telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately 5 times more than that in DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Catalase did not inhibit this oxidative DNA damage, indicating no or little participation of H(2)O(2) in DNA damage. These results indicate that the photoexcited endogenous photosensitizer specifically oxidizes the central guanine of 5'-GGG-3' in telomere sequence to produce 8-oxodG probably through an electron-transfer reaction. It is concluded that the site-specific damage in telomere sequence induced by UVA irradiation may participate in the increase of telomere shortening rate.