首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
The OGG1 gene of Saccharomyces cerevisiae codes for a DNA glycosylase that excises 7,8-dihydro-8- oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Fapy) from damaged DNA. In this paper, we have analysed the substrate specificity and the catalytic mechanism of the Ogg1 protein acting on DNA subtrates containing 8-OxoG residues or apurinic/apyrimidinic (AP) sites. The Ogg1 protein displays a marked preference for DNA duplexes containing 8-OxoG placed opposite a cytosine, the rank order for excision of 8-OxoG and cleavage efficiencies being 8-OxoG/C >8-OxoG/T >>8-OxoG/G and 8-OxoG/A. The cleavage of the DNA strand implies the excision of 8-OxoG followed by abeta-elimination reaction at the 3'-side of the resulting AP site. The Ogg1 protein efficiently cleaves a DNA duplex where a preformed AP site is placed opposite a cytosine (AP/C). In contrast, AP/T, AP/A or AP/G substrates are incised with a very low efficiency. Furthermore, cleavage of 8-OxoG/C or AP/C substrates implies the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre (NaBH4). Therefore, the Ogg1 protein is a eukaryotic DNA glycosylase/AP lyase. Sequence homology searches reveal that Ogg1 probably shares a common ancestor gene with the endonuclease III of Escherichia coli. A consensus sequence indicates a highly conserved lysine residue, K120 of endonuclease III or K241 of Ogg1, respectively. Mutations of K241 to Gln (K241Q) and Arg (K241R) have been obtained after site directed mutagenesis of OGG1. Mutation K241Q completely abolishes DNA glycosylase activity and covalent complex formation in the presence of NaBH4. However, the K241Q mutant still binds DNA duplexes containing 8-OxoG/C. In contrast, K241R mutation results in a catalytically active form of Ogg1. These results strongly suggest that the free amino group of Lys241 is involved in the catalytic mechanism of the Ogg1 protein.  相似文献   

2.
A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.  相似文献   

3.
8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA. The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand. Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions. In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested. We showed that enzymes recognize and specifically bind to DNA duplexes obtained. The mechanism of incision of oxoG by the Fpg and hOGG1 was determined. We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG. In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.  相似文献   

4.
Fapy.dG is produced in DNA as a result of oxidative stress. Under some conditions Fapy.dG is formed in greater yields than 8-oxodG from a common chemical precursor. Recently, Fapy.dG and its C-nucleoside analogue were incorporated in chemically synthesized oligonucleotides at defined sites. Like 8-oxodG, Fapy.dG instructs DNA polymerase to misincorporate dA opposite it in vitro. The interactions of DNA containing Fapy.dG or the nonhydrolyzable analogue with Fpg and MutY are described. Fpg excises Fapy.dG (K(M) = 2.0 nM, k(cat) = 0.14 min(-1)) opposite dC approximately 17-fold more efficiently than when mispaired with dA, which is misinserted by DNA polymerase in vitro. Fpg also prefers to bind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in which the lesion is opposite dA. MutY incises dA when it is opposite Fapy.dG and strongly binds duplexes containing the lesion or beta-C-Fapy.dG. Incision from Fapy.dG.dA is faster than from dG.dA mispairs but slower than from DNA containing 8-oxodG opposite dA. These data demonstrate that Fapy.dG closely resembles the interactions of 8-oxodG with two members of the GO repair pathway in vitro. The similar effects of Fapy.dG and 8-oxodG on DNA polymerase and repair enzymes in vitro raise the question as to whether Fapy.dG elicits similar effects in vivo.  相似文献   

5.
Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.  相似文献   

6.
The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site.  相似文献   

7.
8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.  相似文献   

8.
Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.  相似文献   

9.
PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.  相似文献   

10.
7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.  相似文献   

11.
M Bjors  L Luna  B Johnsen  E Hoff  T Haug  T Rognes    E Seeberg 《The EMBO journal》1997,16(20):6314-6322
The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.  相似文献   

12.
Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are base excision repair enzymes involved in the 8-oxoguanine (oxoG) repair pathway. Specific contacts between these enzymes and DNA phosphate groups play a significant role in DNA-protein interactions. To reveal the phosphates crucial for lesion excision by Fpg and hOGG1, modified DNA duplexes containing pyrophosphate and OEt-substituted pyrophosphate internucleotide (SPI) groups near the oxoG were tested as substrate analogues for both proteins. We have shown that Fpg and hOGG1 recognize and specifically bind the DNA duplexes tested. We have found that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the 8-oxoguanosine (oxodG) and one nucleotide 3' away from it. In contrast, they efficiently incised DNA duplexes bearing the same phosphate modifications 5' to the oxodG and two nucleotides 3' away from the lesion. The effect of these phosphate modifications on the substrate properties of oxoG-containing DNA duplexes is discussed. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or SPI groups immediately 3' to the oxodG or one nucleotide 3' away from it are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a wild-type enzymes bound to oxoG-containing DNA.  相似文献   

13.
The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254?nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (CanR) and reversion to Lys+ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G?·?C→T?·?A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae.  相似文献   

14.
The unusual behavior of the mutation ami36, which generates hyperrecombination in two point crosses, was previously attributed to a localized conversion process changing A/G mispairs into CG pairs. Although the mechanism was found to be dependent on the DNA polymerase I, the specific function responsible for this correction was still unknown. Analysis of the pneumococcal genome sequence has revealed the presence of an open reading frame homologous to the gene mutY of Escherichia coli. The gene mutY encodes an adenine glycosylase active on A/G and A/7,8-dihydro-8-oxoguanine (8-OxoG) mismatches, inducing their repair to CG and C/8-OxoG, respectively. Here we report that disrupting the pneumococcal mutY homologue abolishes the hyperrecombination induced by ami36 and leads to a mutator phenotype specifically enhancing AT-to-CG transversions. The deduced amino acid sequence of the pneumococcal MutY protein reveals the absence of four cysteines, highly conserved in the endonuclease III/MutY glycosylase family, which ligate a [4Fe-4S](2+) cluster. The actual function of this cluster is still intriguing, inasmuch as we show that the pneumococcal gene complements a mutY strain of E. coli.  相似文献   

15.
Bypass of the configurationally stable analogue (beta-C-Fapy x dG) of the formamidopyrimidine lesion derived from 2'-deoxyguanosine oxidation (Fapy x dG) was studied in vitro and in Escherichia coli. The exonuclease deficient Klenow fragment of E. coli DNA polymerase I (Klenow exo(-)) misincorporated dA most frequently opposite beta-C-Fapy x dG, but its efficiency was <0.2% of dC insertion. Klenow exo(-) fidelity was enhanced by the enzyme's high selectivity for extending duplexes only when dC was opposite beta-C-Fapy x dG. The expectations raised by these in vitro data were realized when beta-C-Fapy x dG replication was studied in E. coli by transfecting M13mp7(L2) bacteriophage DNA containing the nucleotide analogue within the lacZ gene in 4 local sequence contexts. The bypass efficiency of beta-C-Fapy x dG varied between 45% and 70% compared to a genome containing only native nucleotides. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay [Delaney, J. C.; Essigmann, J. M. Methods Enzymol.2006, 408, 1]. The levels of mutations could not be distinguished between those observed when genomes containing native nucleotides were replicated, indicating that the mutagenicity of beta-C-Fapy x dG was <1%. These data and previous reports indicate that beta-C-Fapy x dG is a good model of Fapy x dG in E. coli. In addition, these results and the previous report of beta-C-Fapy x dG binding to the base excision repair protein formamidopyrimidine glycosylase suggest that this analogue could be useful as a DNA repair inhibitor.  相似文献   

16.
Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.  相似文献   

17.
The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.  相似文献   

18.
8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.  相似文献   

19.
The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.  相似文献   

20.
The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (CanR) and reversion to Lys+ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G · C→T · A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae. Received: 6 September 1996 / Accepted: 22 October 1996  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号