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S Boiteux

Publications and source records attributed to S Boiteux.

At least 19 recordsLinked to original sources
PubMed2005

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae.

UVA (320-400 nm) radiation constitutes >90% of the environmentally relevant solar UV radiation, and it has been proposed to have a role in skin cancer and aging. Because of the popularity of UVA tanning beds and prolonged periods of sunbathing, the potential deleterious effect of UVA has emerged as a source of concern for public health. Although generally accepted, the impact of DNA damage on the cytotoxic, mutagenic, and carcinogenic effect of UVA radiation remains unclear. In the present study, we investigated the sensitivity of a panel of yeast mutants affected in the processing of DNA damage to the lethal and mutagenic effect of UVA radiation. The data show that none of the major DNA repair pathways, such as base excision repair, nucleotide excision repair, homologous recombination, and postreplication repair, efficiently protect yeast from the lethal action of UVA radiation. In contrast, the results show that the Ogg1 DNA glycosylase efficiently prevents UVA-induced mutagenesis, suggesting the formation of oxidized guanine residues. Furthermore, sequence analysis of UVA-induced canavanine-resistant mutations reveals a bias in favor of GC-->TA events when compared with spontaneous or H(2)O(2)-, UVC-, and gamma-ray- induced canavanine-resistant mutations in the WT strain. Taken together, our data point out a major role of oxidative DNA damage, mostly 7,8-dihydro-8-oxoguanine, in the genotoxicity of UVA radiation in the yeast Saccharomyces cerevisiae. Therefore, the capacity of skin cells to repair 7,8-dihydro-8-oxoguanine may be a key parameter in the mutagenic and carcinogenic effect of UVA radiation in humans.

Aging↗
PubMed2004

Excision by the human methylpurine DNA N-glycosylase of cyanuric acid, a stable and mutagenic oxidation product of 8-oxo-7,8-dihydroguanine.

PURPOSE: 1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-cyanuric acid (cyanuric acid nucleoside or dCa) has been shown to be formed upon exposure of 8-oxo-7,8-dihydroguanine- (8-oxoG) containing oligodeoxyribonucleotides (ODN) to oxidizing agents. When present in DNA, cyanuric acid (Ca) is readily bypassed by Escherichia coli DNA polymerases, which preferentially incorporate 2'-deoxyadenosine-5'-monophosphate (dAMP) opposite to the lesion. Therefore, Ca could be a mutagenic DNA lesion yielding G.C to T.A transversions like 8-oxoG. These results call attention to the potential importance of secondary oxidation products of 8-oxoG. The present study investigates the capability of several DNA N-glycosylases to remove the Ca lesion in DNA. MATERIALS AND METHODS: A site-specifically modified 22-mer ODN containing a single Ca residue was hybridized with complementary sequences yielding four DNA duplexes harbouring Ca opposite each of the regular DNA bases. The four Ca.N duplexes were used as substrates for nine DNA N-glycosylases from bacterial, yeast or human origin. RESULTS: The results show that the human methylpurine DNA N-glycosylase (Mpg) can remove Ca from DNA duplexes. Interestingly, oxidized base-specific DNA N-glycosylases, Fpg, Nth, Ntg1, Ntg2, Ogg1, hNth1 and hOgg1, cannot repair Ca in DNA. Furthermore, the removal of Ca by Mpg varied markedly depending on the opposite DNA base, the rank being Ca.C=Ca.T>Ca.G=Ca.A. CONCLUSIONS: 8-OxoG-derived lesions in DNA such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), oxaluric acid (Oa), oxazolone (Oz) and Ca are substrates of base excision repair DNA N-glycosylases. Most of them, Sp, Gh, Oa and Oz, are substrates of the oxidized bases-specific enzymes such as Nth or Fpg. In contrast, Ca is substrate of the human methylpurine DNA N-glycosylase (Mpg).

Binding Sites↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2001

XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions.

The major human AP endonuclease APE1 (HAP1, APEX, Ref1) initiates the repair of abasic sites generated either spontaneously, from attack of bases by free radicals, or during the course of the repair of damaged bases. APE1 therefore plays a central role in the base excision repair (BER) pathway. We report here that XRCC1, another essential protein involved in the maintenance of genome stability, physically interacts with APE1 and stimulates its enzymatic activities. A truncated form of APE1, lacking the first 35 amino acids, although catalytically proficient, loses the affinity for XRCC1 and is not stimulated by XRCC1. Chinese ovary cell lines mutated in XRCC1 have a diminished capacity to initiate the repair of AP sites. This defect is compensated by the expression of XRCC1. XRCC1, acting as both a scaffold and a modulator of the different activities involved in BER, would provide a physical link between the incision and sealing steps of the AP site repair process. The interaction described extends the coordinating role of XRCC1 to the initial step of the repair of DNA abasic sites.

Animals↗
PubMed2001

Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGG1 protein.

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step.

The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.

Amino Acid Substitution↗
PubMed2001

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein.

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G.C-->T.A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5' or 3' opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3'- and 5'-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.

Apurinic Acid↗
PubMed2001

Synergism between base excision repair, mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.

Alleles↗
PubMed2001

Repair of 8-oxoguanine and Ogg1-incised apurinic sites in a CHO cell line.

The repair mechanisms involved in the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) in damaged DNA have been investigated using cell-free extracts or purified proteins. However, in vivo repair assays are required to further dissect mechanisms involved in the repair of 8-oxoG in the cellular context. In this study, we analyzed the removal of 8-oxoG from plasmids that contain a single 8-oxoG.C base pair in a sequence that can be transcribed (TS) or nontranscribed (NTS) in a chinese hamster ovary (CHO) cell line. The results show that 8-oxoG located in a TS is removed faster than in a NTS, indicating transcription-coupled repair (TCR) of 8-oxoG in rodent cells. The results also show that CHO cells efficiently repair DNA molecules that contain an Ogg1-incised AP site, which is the first intermediate in the course of base excision repair of 8-oxoG.

Animals↗
PubMed2000

Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines.

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.

Amino Acid Sequence↗
PubMed2000

Characterization of the hOGG1 promoter and its expression during the cell cycle.

The human OGG1 gene codes for a 38kD protein with an antimutator activity related to its capacity to excise the mutagenic base 8-OH-Guanine from DNA. Mutant forms of this gene have been found in lung and kidney tumors. The determination of the start of transcription allowed the definition of the promoter sequences for the gene. By transient transfection and a luciferase reporter assay a 135 base pair region immediately upstream of the transcription start is shown to have full promoter activity. Two CpG islands and an Alu repeat were identified within the promoter and the 5' sequences of the transcribed region. The lack of TATA or CAAT boxes suggests that OGG1 is a housekeeping gene. Consistently, its expression, measured as the transcription from the promoter or as the enzymatic activity in cultured fibroblast cell lines, does not vary during the cell cycle.

Base Sequence↗
PubMed2000

Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity.

We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins.

Bacterial Proteins↗
PubMed2000

Alterations of the DNA repair gene OGG1 in human clear cell carcinomas of the kidney.

The OGG1 gene, which codes for a DNA repair protein with antimutator activity, is located on chromosome 3p25, a frequent site of allelic deletions in many types of human tumors, including renal clear cell cancers. We present the analysis of 99 renal tumors for alterations in the OGG1 gene to determine its association with tumorigenesis. Loss of heterozygosity in the 3p25 region was found for 85% of the informative cases. We detected somatic missense mutations of the OGG1 gene in 4 of the 99 tumor samples. Biochemical analysis of the mutant proteins revealed that a substitution at codon 46 impairs the enzymatic activity. We also describe the occurrence of several polymorphisms as well as aberrantly spliced OGG1 transcripts.

Adenocarcinoma, Clear Cell↗
PubMed2000

Transcription coupled repair of 8-oxoguanine in murine cells: the ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences.

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7, 8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1(-/-) null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG.C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1(-/-) MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1(-/-) cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

Animals↗
PubMed2000

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases.

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.

Amino Acid Motifs↗
PubMed2000

The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis.

A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as byproducts of cell metabolism. Among DNA damages induced by ROS, 8-hydroxyguanine (8-OH-G) is certainly the product that has retained most of the attention in the past few years. The biological relevance of 8-OH-G in DNA has been unveiled by the study of Escherichia coli and Saccharomyces cerevisiae genes involved in the neutralization of the mutagenic effects of 8-OH-G. These genes, fpg and mutY for E. coli and OGG1 for yeast, code for DNA glycosylases. Inactivation of any of those genes leads to a spontaneous mutator phenotype, characterized by the increase in GC to TA transversions. In yeast, the OGG1 gene encodes a DNA glycosylase/AP lyase that excises 8-OH-G from DNA. In human cells, the OGG1 gene is localized on chromosome 3p25 and encodes two forms of hOgg1 protein which result from an alternative splicing of a single messenger RNA. The alpha-hOgg1 protein has a nuclear localization whereas the beta-hOgg1 is targeted to the mitochondrion. Biochemical studies on the alpha-hOgg1 protein show that it is a DNA glycosylase/AP lyase that excises 8-OH-G and Fapy-G from gamma-irradiated DNA. Several approaches have been used to study the biological role of OGG1 in mammalian cells, ranging from its overexpression in cell lines to the generation of homozygous ogg1-/- null mice. Furthermore, to explore a possible role in the prevention of cancer, the cDNA coding for alpha-hOgg1 has been sequenced in human tumors. All these results point to 8-OH-G as an endogenous source of mutations in eukaryotes and to its likely involvement in the process of carcinogenesis. A review of the recent literature on the mammalian Ogg1 proteins, the main repair system involved in the elimination of this mutagenic lesion, is presented.

Aging↗