Biomedical subjects

S Boiteux

Publications and source records attributed to S Boiteux.

At least 55 records · Page 3Linked to original sources

PubMed1997

Inactivation of OGG1 increases the incidence of G . C-->T . A transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells.

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 x C-->T x 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.

Base Sequence↗

PubMed1997

Repair of oxidized DNA bases in the yeast Saccharomyces cerevisiae.

An essential requirement for all organisms is to maintain its genomic integrity. Failure to do so, in multicellular organisms such as man, can lead to degenerative pathologies such as cancer and aging. Indeed, a very low spontaneous mutation rate is observed in eukaryotes, suggesting either an inherent stability of the genome or efficient DNA repair mechanisms. In fact, DNA is subjected to unceasing attacks by a variety of endogenous and environmental reactive chemical species yielding a multiplicity of DNA damage, the deleterious action of which is counteracted by efficient repair enzymes. Reactive oxygen species formed in cell as by-products of normal metabolism are probably the major source of endogenous DNA damage. Amongst oxidative damage, base modifications constitute an important class of lesions whose lethal or mutagenic action has been established. Oxidatively damaged DNA bases are mostly repaired by the base excision repair pathway (BER) in prokaryotes and eukaryotes. However, the nucleotide excision repair pathway (NER) may also play a role in the repair of some oxidized bases in DNA. Here, we describe repair pathways implicated in the removal of oxidized bases in Saccharomyces cerevisiae. Yeast is a simple organism that can be used as a paradigm for DNA repair in all eukaryotic cells. S cerevisiae possesses three DNA glycosylases that catalyze the excision of oxidized bases from damaged DNA: the Ogg1, Ntg1 and Ntg2 proteins. The aim of this review is to summarize recent findings dealing with the formation, the biological consequences and the repair of oxidized DNA bases in S cerevisiae.

Amino Acid Sequence↗

PubMed1997

Analysis of the mutations induced in the E. coli lac Z gene by a psoralen analog.

A psoralen in which intracyclic oxygen atoms were replaced by sulfur (7H-thieno [3,2-g]-[1]-[benzothiopyran-7-one) [PSO(S-S)]) was recently synthesized and its photobiological properties were investigated. M13mp19 DNA photosensitization mediated by PSO (S-S) followed by transfection into competent E. coli gave rise to a very low phage progeny showing the high aptitude of this compound to modify DNA. In order to characterize the role of oxidative damages in the photosensitized reaction mediated by PSO(S-S), plasmid bearing the gene encoding the formamidopyrimidine DNA glycosylase (Fpg) under the control of the inducible lac Z promoter was transfected in E. coli. Overexpression of Fpg was induced by addition of isopropyl-beta-D-thio-galactopyranoside (IPTG) to the cells and monitored by western blot analysis. Fpg overexpression did not influence the rate of M13mp19 DNA photoinactivation by PSO(S-S) neither the mutation frequency measured by the expression of beta-galactosidase encoded by the lac Z gene beared by M13mp19. Analysis of the mutation patterns recorded with or without Fpg overexpression showed that several G to T transversions due to oxidative damages were repaired by Fpg. These data show that oxidative DNA damages generated during PSO(S-S) photosensitization have only limited biological implications measured in terms of DNA photoinactivation.

Bacteriophage M13↗

PubMed1997

Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA.

The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac181. Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg. The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites. Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions. To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisiae. In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain. However, the transforming efficiency was not affected in Fpg-deficient strains. In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14::URA3 strain. The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities. The results show that the yeast strain defective in the nucleotide excision repair pathway (rad14::URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain. It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14::URA3 mutant only exhibits a minor oxygen dependence. To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro- and eukaryotic cells by the nucleotide excision repair pathway.

Carbon-Oxygen Lyases↗

PubMed1997

[Repair of oxidized guanine in mammals: OGG1 genes].

This paper reviews the present state of the studies on the repair of a major oxydative lesion on DNA, the 8-oxo-guanine (8-OxoG). This modified base has been proved to be highly mutagenic and therefore implicated in the ethiology of several pathologies. The cloning of the yeast OGG1 gene, a functional homolog of the fpg from bacteria, allowed the isolation of the mammalian homologs. These genes code for 8-OxoG DNA glycosylases/lyases, whose biochemical properties are consistent with their postulated role as the main defence against the genetic instability induced by the presence of 8-OxoG in DNA. This, together with the mutator phenotype of the yeast ogg1 mutant strains, make of the human OGG1 a candidate for a cancer predisposition gene. The localization of this gene to chromosome 3p and other evidences discussed in this paper indicate that OGG1 could be a tumor suppressor gene implicated in lung cancer.

Animals↗

PubMed1996

Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.

A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.

Amino Acid Sequence↗

PubMed1996

UV-induced endonuclease III-sensitive sites at the mating type loci in Saccharomyces cerevisiae are repaired by nucleotide excision repair: RAD7 and RAD16 are not required for their removal from HML alpha.

Ultraviolet irradiation of DNA induces cyclobutane pyrimidine dimers (CPDs) 6-4'-(pyrimidine 2'-one) pyrimidines and pyrimidine hydrates. The dimer is the major photoproduct, and is specifically recognized by endonuclease V of phage T4. Pyrimidine hydrates represent a small fraction of the total photoproducts, and are substrates for endonuclease III of Escherichia coli. We used these enzymes to follow the fate of their substrates in the mating type loci of Saccharomyces cerevisiae. In a RAD strain, CPSs in the transcriptionally active MAT alpha locus are preferentially repaired relative to the inactive HML alpha locus, whilst repair of endonuclease III-sensitive sites is not preferential. The rad1, 2, 3 and 4 mutants, which lack factors that are essential for the incision step of nucleotide excision repair (NER), repair neither CPDs nor endonuclease III-sensitive sites, clearly showing that these lesions are repaired by by NER pathway. Previously it had been shown that the products of the RAD7 and RAD16 genes are required for the NER of CPDs from the HML alpha locus. We show that, in the same locus, these gene products are not needed for removal of endonuclease III-sensitive sites by the same mechanism. This indicates that the components required for NER differ depending on either the type of lesion encountered or on the specific location of the lesion within the genome.

Adenosine Triphosphatases↗

PubMed1996

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant for RAD14, the Saccharomyces cerevisiae homologue of the human gene defective in XPA patients.

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in the MAT alpha and HML alpha loci of Saccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in a rad14-2 point mutant and in a rad14 deletion mutant. The RAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III site (i.e. as wild type), but no removal of CPDs at the MAT alpha and HML alpha loci. Similar experiments were undertaken using the recently created rad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired in MATa or HMRa. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that in the genome overall, repair of 6-4 photoproducts was less impaired that repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that the rad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.

DNA Repair↗

PubMed1996

Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair.

An Arabidopsis thaliana cDNA was isolated by complementation of the Escherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways. This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535. It also partially corrects the UV-sensitive phenotype of E. coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated lambda phage. It has an insert of ca. 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa). This cDNA presents a high homology to a stress related gene from two species of Fusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway, THI4, from Saccharomyces cerevisiae and nmt2 from Schizosaccharomyces pombe. The Arabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases. The auxotrophy for thiamine in the yeast thi4::URA3 disruption strain is complemented by the Arabidopsis gene. Thus, the cloned gene, named thi1, is likely to function in the biosynthesis of thiamine in plants. The data presented in this work indicate that thi1 may also be involved in DNA damage tolerance in plant cells.

Amino Acid Sequence↗

PubMed1995

Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.

The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.

Base Sequence↗

PubMed1995

HU protein of Escherichia coli binds specifically to DNA that contains single-strand breaks or gaps.

In this study, we have identified a protein in Escherichia coli that specifically binds to double-stranded DNA containing a single-stranded gap of one nucleotide. The gap-DNA binding (GDB) protein was purified to apparent homogeneity. The analysis of the amino-terminal sequencing of the GDB protein shows two closely related sequences we identify as the alpha and beta subunits of the HU protein. Furthermore, the GDB protein is not detected in the crude extract of an E. coli double mutant strain hupA hupB that has no functional HU protein. These results led us to identify the GDB protein as the HU protein. HU binds strongly to double-stranded 30-mer oligonucleotides containing a nick or a single-stranded gap of one or two nucleotides. Apparent dissociation constants were measured for these various DNA duplexes using a gel retardation assay. The KD(app) values were 8 nM for the 30-mer duplex that contains a nick and 4 and 2 nM for those that contain a 1-or a 2-nucleotide gap, respectively. The affinity of HU for these ligands is at least 100-fold higher than for the same 30-mer DNA duplex without nick or gap. Other single-stranded breaks or gaps, which are intermediate products in the repair of abasic sites after incision by the Fpg, Nth, or Nfo proteins, are also preferentially bound by the HU protein. Due to specific binding to DNA strand breaks, HU may play a role in replication, recombination, and repair.

Amino Acid Sequence↗

PubMed1995

Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein.

The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 has been cloned, characterized and sequenced. The fpg-L gene is composed of 819 bp encoding a protein of 31.3 kDa (Fpg-L). The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identity with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodies against Fpg-E react with the Fpg-L protein. The Fpg-L protein was purified to apparent homogeneity from the overproducing E. coli strain BH410 hosting plasmid pVE1064, which carries fpg-L under the control of the E. coli lac promoter. In its active form, Fpg-L is a 30 kDa monomeric enzyme with a measured isoelectric point of 9.0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxyterminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH). The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of the fpg-L gene in fpg and mutY mutants of E. coli suppresses their spontaneous GC-->TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conversation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.

Amino Acid Sequence↗

PubMed1994

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.

Base Sequence↗

PubMed1994

Recognition of oxidized abasic sites by repair endonucleases.

The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.

Bleomycin↗

PubMed1994

Visible light generates oxidative DNA base modifications in high excess of strand breaks in mammalian cells.

The DNA damage induced by visible light in L1210 mouse leukaemia cells was analysed by an alkaline elution assay with specific repair endonucleases. DNA single-strand breaks and DNA modifications sensitive to FPG protein (formamidopyrimidine-DNA glycosylase), endonuclease III and exonuclease III were quantified in parallel. The light-induced cellular DNA damage was found to consist of many base modifications sensitive to FPG protein, which most probably are predominantly 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) residues. Base modifications sensitive to endonuclease III are virtually absent. The yield of the FPG-sensitive base modifications is 10-fold higher than that of single-strand breaks plus AP sites (sites of base loss). The described ratios of the various modifications indicate that the damage most probably results from a reaction of DNA with singlet oxygen (type II reaction) or directly with an excited endogenous photosensitizer (type I reaction) and is not mediated by hydroxyl radicals. Experiments with cut-off filters indicate that wavelengths between 400 and 500 nm are responsible for most of the modifications. The FPG-sensitive base modifications are repaired efficiently (t1/2 approximately 1 h at 37 degrees C). This is perhaps why the light-induced DNA damage is apparently associated with only low mutagenicity.

Animals↗

PubMed1994

Fpg protein protects Escherichia coli K-12 from mutation induction by the carcinogen 4-nitroquinoline 1-oxide.

This paper investigates the role of the fpg gene product in protecting Escherichia coli cells against the lethal and mutagenic effects of 4-nitroquinoline 1-oxide (4NQO). To this end, the araD81 mutation which make the cells sensitive to L-arabinose was combined with an fpg-1::Knr allele, in either an uvrA+ or uvrA-, umuC+ or umuC-, genetic background. Mutation induction was monitored by selecting forward mutations to L-arabinose resistance (Arar). The formamidopyrimidine-DNA glycosylase (Fpg protein) protected bacteria from 4NQO-induced mutagenesis since Fpg- defective cells showed greater Arar mutation induction than fpg+ bacteria did. This was confirmed since the increased sensitivity of the fpg- cells to mutagenesis by 4NQO was suppressed when the Fpg protein was overproduced by placing the fpg gene in a multicopy plasmid vector. The fpg- mutation had no detectable influence on 4NQO mutagenesis in a uvrA- genetic background, but its effect was magnified in umuC- cells. No influence on cell survival was observed after 4NQO treatment. Our data suggest that 8-hydroxyguanine, a non-lethal, non-bulky and directly miscoding lesion, might be responsible for the detected influence of Fpg protein expression on mutation induction by 4NQO. This is in agreement with the reported in vivo formation of 8-hydroxyguanine in cellular DNA after 4NQO exposure. The increased 4NQO-induction of GC to TA transversions on fpg- bacteria further support such a possibility. This work reinforces the role of Fpg protein in the bacterial defense against the mutagenicity by genotoxic agents.

4-Nitroquinoline-1-oxide↗

PubMed1993

Substrate specificity of the Escherichia coli endonuclease III: excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals.

The excision of modified bases from DNA by Escherichia coli endonuclease III was investigated. Modified bases were produced in DNA by exposure of dilute buffered solutions of DNA to ionizing radiation under oxic or anoxic conditions. The technique of gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 16 pyrimidine- and purine-derived DNA lesions. DNA substrates were incubated either with the native enzyme or with the heat-inactivated enzyme. Subsequently, DNA was precipitated. Pellets were analyzed by GC/MS after hydrolysis and derivatization. Supernatant fractions were analyzed after derivatization without hydrolysis. The results provided unequivocal evidence for the excision by E. coli endonuclease III of a number of thymine- and cytosine-derived lesions from DNA. These were 5,6-dihydrothymine, 5-hydroxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. None of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonuclease III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repair enzyme in the context of a large number of pyrimidine- and purine-derived lesions in DNA.

Animals↗

PubMed1993

Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins.

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.

Amino Acid Sequence↗