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Raghuvir K Arni - One of the best experts on this subject based on the ideXlab platform.
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chemical and thermal influence of the 4fe 4s 2 cluster of a g specific adenine Glycosylase from corynebacterium pseudotuberculosis
Biochimica et Biophysica Acta, 2015Co-Authors: Raphael J Eberle, Monika A Coronado, Icaro Putinhon Caruso, Debora De Oliveira Lopes, Anderson Miyoshi, Vasco Azevedo, Raghuvir K ArniAbstract:Abstract The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA Glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe–4S]2 + cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe–4S]2 + cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe–4S]2 + cluster. UV–vis and fluorescence spectroscopy results demonstrate that between pH 3.0 and 4.0 the integrity of the [4Fe–4S]2 + cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45 °C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.
Groisman Regina - One of the best experts on this subject based on the ideXlab platform.
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Aberrant repair initiated by the adenine-DNA Glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli
'PeerJ', 2018Co-Authors: Zutterling Caroline, Mursalimov Aibek, Talhaoui Ibtissam, Koshenov Zhanat, Akishev Zhiger, Bissenbaev Amangeldy, Mazon Gerard, Geacintov Nicolas, Gasparutto Didier, Groisman ReginaAbstract:International audienceBackground: DNA repair is essential to counteract damage to DNA induced by endo-and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA Glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA Glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T/C•G transversions. Furthermore, it has been demonstrated that human MutYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. Results: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli MutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C/T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. Discussion: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damag
Regina Groisman - One of the best experts on this subject based on the ideXlab platform.
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Aberrant repair initiated by the adenine-DNA Glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli
PeerJ, 2018Co-Authors: Caroline Zutterling, Aibek Mursalimov, Ibtissam Talhaoui, Zhanat Koshenov, Zhiger Akishev, Amangeldy Bissenbaev, Gerard Mazon, Nicolas Geacintov, Didier Gasparutto, Regina GroismanAbstract:Background: DNA repair is essential to counteract damage to DNA induced by endo-and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA Glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA Glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T/C•G transversions. Furthermore, it has been demonstrated that human MutYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. Results: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli MutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C/T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. Discussion: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage
Raphael J Eberle - One of the best experts on this subject based on the ideXlab platform.
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chemical and thermal influence of the 4fe 4s 2 cluster of a g specific adenine Glycosylase from corynebacterium pseudotuberculosis
Biochimica et Biophysica Acta, 2015Co-Authors: Raphael J Eberle, Monika A Coronado, Icaro Putinhon Caruso, Debora De Oliveira Lopes, Anderson Miyoshi, Vasco Azevedo, Raghuvir K ArniAbstract:Abstract The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA Glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe–4S]2 + cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe–4S]2 + cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe–4S]2 + cluster. UV–vis and fluorescence spectroscopy results demonstrate that between pH 3.0 and 4.0 the integrity of the [4Fe–4S]2 + cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45 °C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.
Tone Tonjum - One of the best experts on this subject based on the ideXlab platform.
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antimutator role of DNA Glycosylase MutY in pathogenic neisseria species
Journal of Bacteriology, 2005Co-Authors: Tonje Davidsen, Magnar Bjoras, Erling Seeberg, Tone TonjumAbstract:Infections caused by Neisseria meningitidis (the meningococcus; MC) and Neisseria gonorrhoeae (the gonococcus; GC), pathogenic members of the genus Neisseria, are associated with significant morbidity and mortality in their exclusive human host. MC and GC residing on mucosal surfaces are exposed to DNA-damaging agents from a potent immune system and also suffer genotoxic stress from endogenous sources, predisposing factors for mutations (29, 36). Mutator strains exhibit an increased spontaneous mutation rate compared to those commonly found in the corresponding wild-type species (17). Such a phenotype is often caused by heritable changes in components of the methyl-directed mismatch repair (MMR) pathway engaged in postreplication repair. However, Richardson et al. (41) demonstrated that only 39% of MC strains exhibiting elevated spontaneous mutation rates could be fully or partially complemented with wild-type mutS or mutL alleles and thus directly linked to defects in the MMR system. Conflicting evidence exists on the association of Dam methylase variants causing hypermutable neisserial strains with enhanced phase-variable capsule switching (4, 21, 40). Clearly, mechanisms other than MMR are implicated in MC mutator phenotypes. Associations between hypermutation and defects in MMR have not yet been reported in the close relative GC. One of the most frequent forms of oxidative DNA damage is the oxidation product of guanine, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8oxoG) (8). The base excision repair (BER) pathway is probably the cell's major line of defense against the deleterious effects of such DNA damage (45). BER involves the release of modified base residues from DNA by DNA Glycosylases that leave abasic (AP) sites in the DNA. The AP site may be further cleaved by an AP-lyase activity inherent of many DNA Glycosylases or by an AP endonuclease, leaving a strand break with a deoxyribose phosphate residue at the 3′ end or 5′ end, respectively. DNA Glycosylases exist in all species so far investigated, confirming a conserved and important role for BER in protection against DNA damage. The DNA Glycosylase MutY is an atypical Glycosylase in the sense that it removes a normal base, adenine, from DNA when it is mispaired with 8oxoG, thereby preventing CG→AT transversions (32). 8oxoG mispairs are formed in vivo during DNA replication by two mechanisms, either incorporation of an adenine nucleotide opposite an 8oxoG derived from the direct oxidation in the template strand (8) or misincorporation of an 8oxoG that results from oxidation of GTP in the nucleotide pool (27). In Escherichia coli, MutY acts together with formamidopyrimidine DNA Glycosylase (Fpg/MutM) and MutT, comprising the 8oxoG (GO) system, to prevent fixation of mutations caused by 8oxoG (30, 31). Fpg removes 8oxoG when paired with cytosine (48). MutT is a hydrolase that converts 8oxodGTP to 8oxodGMP when present in the nucleotide pool (27), thereby preventing 8oxoG from being misincorporated during replication. Inactivation of each of these genes individually in E. coli confers a mutator phenotype, and E. coli fpg, MutY, and mutT mutants have been reported as weak, moderate, and strong mutators, respectively (13, 30). MutY belongs to a superfamily of DNA repair proteins hallmarked by a helix-hairpin-helix (HhH) motif involved in nonsequence-specific DNA binding (46). The HhH family includes other DNA Glycosylases, such as endonuclease III (Nth) and DNA-3-methyladenine (AlkA). An evolutionary analysis of the HhH superfamily of DNA repair Glycosylases performed by Denver et al. shows that MutY is present in most bacteria, many eukaryotes, and nearly 50% of the archaea investigated (9). The crystal structure of E. coli MutY has been solved (15, 19), and the gene has been cloned and characterized in a range of species, including mammals (23, 24, 28, 43). However, none of these species are directly comparable with MC. In this work, we report the characterization of the neisserial MutY gene and of its gene product, which induces a hypermutable phenotype in both MC and GC when inactivated. The protein encoded by the MutY gene has been overexpressed, purified to homogeneity, and assessed for its activities and substrate specificity. Furthermore, functional phenotypes of MC and GC MutY null mutants were assessed.