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Leona D Samson - One of the best experts on this subject based on the ideXlab platform.
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mammalian 3 methyladenine DNA Glycosylase protects against the toxicity and clastogenicity of certain chemotherapeutic DNA cross linking agents
Cancer Research, 1998Co-Authors: James M. Allan, Michael D Wyatt, Andrew J Dreslin, Maria Tomasz, Bevin P Engelward, Leona D SamsonAbstract:DNA repair status is recognized as an important determinant of the clinical efficacy of cancer chemotherapy. To assess the role that a mammalian DNA Glycosylase plays in modulating the toxicity and clastogenicity of the chemotherapeutic DNA cross-linking alkylating agents, we compared the sensitivity of wild-type murine cells to that of isogenic cells bearing homozygous null mutations in the 3-methyladenine DNA Glycosylase gene ( Aag ). We show that Aag protects against the toxic and clastogenic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C (MMC), as measured by cell killing, sister chromatid exchange, and chromosome aberrations. This protection is accompanied by suppression of apoptosis and a slightly reduced p53 response. Our results identify 3-methyladenine DNA Glycosylase-initiated base excision repair as a potentially important determinant of the clinical efficacy and, possibly, the carcinogenicity of these widely used chemotherapeutic agents. However, Aag does not contribute significantly to protection against the toxic and clastogenic effects of several chemotherapeutic nitrogen mustards (namely, mechlorethamine, melphalan, and chlorambucil), at least in the mouse embryonic stem cells used here. We also compare the Aag null phenotype with the Fanconi anemia phenotype, a human disorder characterized by cellular hypersensitivity to DNA cross-linking agents, including MMC. Although Aag null cells are sensitive to MMC-induced growth delay and cell cycle arrest, their sensitivity is modest compared to that of Fanconi anemia cells.
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repair deficient 3 methyladenine DNA Glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, Andrew J Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:Abstract In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA Glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA Glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA Glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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repair deficient 3 methyladenine DNA Glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, Andrew J Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA Glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA Glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA Glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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cloning and characterization of a mouse 3 methyladenine 7 methl guanine methylguanine DNA Glycosylase cDNA whose gene maps to chromosme 11
Carcinogenesis, 1993Co-Authors: Bevin P Engelward, Michael S. Boosalis, Zuoming Deng, Beiru J Chen, Michael J Siciliano, Leona D SamsonAbstract:: In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions by DNA Glycosylases prevents alkylation induced cell death. We described previously the isolation of a human 3MeA DNA Glycosylase (AAG) cDNA that maps to chromosome 16 and hybridizes to specific genomic DNA fragments from a number of mammals, including mouse. As a first step in the generation of a 3MeA DNA Glycosylase deficient mouse by homologous replacement in embryonic stem cells, we have cloned the mouse 3MeA DNA Glycosylase cDNA. The cloned 1095 base pair cDNA contains a complete 333 amino acid open reading frame that predicts a 36.5 kDa protein and hybridizes to a 1.5 kb mRNA transcript. Mouse 3MeA DNA Glycosylase (Aag) transcript levels vary by up to 21 fold among tissues, being highest in the testes and lowest in the heart. The Aag cDNA encodes a Glycosylase able to release 3MeA, 7-methylguanine (7MeG) and 3-methylguanine (3MeG) from alkylated DNA. The expression of Aag in E. coli provides substantial resistance against killing by methylating agents, but, unlike its E. coli counterparts, the Aag Glycosylase fails to protect against killing by ethylating and propylating agents. A 232 amino acid stretch of the predicted mouse protein shares extensive amino acid identity with rat (93%) and human (83%) 3MeA DNA Glycosylases and we observe that all three mammalian Glycosylases have a bipartite nuclear localization signal. The Aag gene maps to mouse chromosome 11, suggesting a segment of conserved synteny between mouse chromosome 11 and human chromosome 16, which bears the human 3MeA DNA Glycosylase gene. Cloning the mouse 3MeA DNA Glycosylase cDNA is a step toward understanding the role of this DNA repair enzyme in mammals.
Bevin P Engelward - One of the best experts on this subject based on the ideXlab platform.
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in vivo repair of methylation damage in aag 3 methyladenine DNA Glycosylase null mouse cells
Nucleic Acids Research, 2000Co-Authors: Stephen A Smith, Bevin P EngelwardAbstract:3-Methyladenine (3MeA) DNA Glycosylases initiate base excision repair by removing 3MeA. These Glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA Glycosylase (also known as the Mpg, APNG or ANPG DNA Glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in Aag–/– cells, 3MeA lesions disappear from the genome slightly faster than would be expected by spontaneous depurination alone, suggesting that there may be residual repair of 3MeA. However, repair of 3MeA is at least 10 times slower in Aag–/– cells than in Aag+/+ cells. Consequently, 24 h after exposure to [3H]MNU, 30% of the original 3MeA burden is intact in Aag–/– cells, while 3MeA is undetectable in Aag+/+ cells. Thus, Aag is the major DNA Glycosylase for 3MeA repair. We also investigated the in vivo repair kinetics of another Aag substrate, 7-methylguanine. Surprisingly, 7-methylguanine is removed equally efficiently in Aag+/+ and Aag–/– cells, suggesting that another DNA Glycosylase acts on lesions previously thought to be repaired by Aag.
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mammalian 3 methyladenine DNA Glycosylase protects against the toxicity and clastogenicity of certain chemotherapeutic DNA cross linking agents
Cancer Research, 1998Co-Authors: James M. Allan, Michael D Wyatt, Andrew J Dreslin, Maria Tomasz, Bevin P Engelward, Leona D SamsonAbstract:DNA repair status is recognized as an important determinant of the clinical efficacy of cancer chemotherapy. To assess the role that a mammalian DNA Glycosylase plays in modulating the toxicity and clastogenicity of the chemotherapeutic DNA cross-linking alkylating agents, we compared the sensitivity of wild-type murine cells to that of isogenic cells bearing homozygous null mutations in the 3-methyladenine DNA Glycosylase gene ( Aag ). We show that Aag protects against the toxic and clastogenic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C (MMC), as measured by cell killing, sister chromatid exchange, and chromosome aberrations. This protection is accompanied by suppression of apoptosis and a slightly reduced p53 response. Our results identify 3-methyladenine DNA Glycosylase-initiated base excision repair as a potentially important determinant of the clinical efficacy and, possibly, the carcinogenicity of these widely used chemotherapeutic agents. However, Aag does not contribute significantly to protection against the toxic and clastogenic effects of several chemotherapeutic nitrogen mustards (namely, mechlorethamine, melphalan, and chlorambucil), at least in the mouse embryonic stem cells used here. We also compare the Aag null phenotype with the Fanconi anemia phenotype, a human disorder characterized by cellular hypersensitivity to DNA cross-linking agents, including MMC. Although Aag null cells are sensitive to MMC-induced growth delay and cell cycle arrest, their sensitivity is modest compared to that of Fanconi anemia cells.
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repair deficient 3 methyladenine DNA Glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, Andrew J Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:Abstract In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA Glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA Glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA Glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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repair deficient 3 methyladenine DNA Glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, Andrew J Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA Glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA Glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA Glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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cloning and characterization of a mouse 3 methyladenine 7 methl guanine methylguanine DNA Glycosylase cDNA whose gene maps to chromosme 11
Carcinogenesis, 1993Co-Authors: Bevin P Engelward, Michael S. Boosalis, Zuoming Deng, Beiru J Chen, Michael J Siciliano, Leona D SamsonAbstract:: In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions by DNA Glycosylases prevents alkylation induced cell death. We described previously the isolation of a human 3MeA DNA Glycosylase (AAG) cDNA that maps to chromosome 16 and hybridizes to specific genomic DNA fragments from a number of mammals, including mouse. As a first step in the generation of a 3MeA DNA Glycosylase deficient mouse by homologous replacement in embryonic stem cells, we have cloned the mouse 3MeA DNA Glycosylase cDNA. The cloned 1095 base pair cDNA contains a complete 333 amino acid open reading frame that predicts a 36.5 kDa protein and hybridizes to a 1.5 kb mRNA transcript. Mouse 3MeA DNA Glycosylase (Aag) transcript levels vary by up to 21 fold among tissues, being highest in the testes and lowest in the heart. The Aag cDNA encodes a Glycosylase able to release 3MeA, 7-methylguanine (7MeG) and 3-methylguanine (3MeG) from alkylated DNA. The expression of Aag in E. coli provides substantial resistance against killing by methylating agents, but, unlike its E. coli counterparts, the Aag Glycosylase fails to protect against killing by ethylating and propylating agents. A 232 amino acid stretch of the predicted mouse protein shares extensive amino acid identity with rat (93%) and human (83%) 3MeA DNA Glycosylases and we observe that all three mammalian Glycosylases have a bipartite nuclear localization signal. The Aag gene maps to mouse chromosome 11, suggesting a segment of conserved synteny between mouse chromosome 11 and human chromosome 16, which bears the human 3MeA DNA Glycosylase gene. Cloning the mouse 3MeA DNA Glycosylase cDNA is a step toward understanding the role of this DNA repair enzyme in mammals.
Tomas Lindahl - One of the best experts on this subject based on the ideXlab platform.
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excision of deaminated cytosine from the vertebrate genome role of the smug1 uracil DNA Glycosylase
The EMBO Journal, 2001Co-Authors: Hilde Nilsen, Karl A Haushalter, Peter Robins, Deborah E Barnes, Gregory L Verdine, Tomas LindahlAbstract:Gene‐targeted mice deficient in the evolutionarily conserved uracil–DNA Glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung − mutants. A complementary uracil–DNA Glycosylase activity detected in ung −/− murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo . Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil–DNA Glycosylase in UNG‐deficient mice. SMUG1 is present at similar levels in cell nuclei of non‐proliferating and proliferating tissues, indicating a replication‐ independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti‐mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.
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uracil DNA Glycosylase ung deficient mice reveal a primary role of the enzyme during DNA replication
Molecular Cell, 2000Co-Authors: Hilde Nilsen, Peter Robins, Geir Slupphaug, Hans E Krokan, Ian Rosewell, Camilla Skjelbred, Sonja Andersen, Graham Daly, Tomas LindahlAbstract:Abstract Gene-targeted knockout mice have been generated lacking the major uracil-DNA Glycosylase, UNG. In contrast to ung − mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung −/− nuclei and an elevated steady-state level of uracil in DNA in dividing ung −/− cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA Glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.
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distinct repair activities of human 7 8 dihydro 8 oxoguanine DNA Glycosylase and formamidopyrimidine DNA Glycosylase for formamidopyrimidine and 7 8 dihydro 8 oxoguanine
Journal of Biological Chemistry, 2000Co-Authors: Kenjiro Asagoshi, Yoshiaki Monden, Hiroaki Terato, Yoshihiko Ohyama, Tsuyoshi Arai, Takao Yamada, Hiroyuki Aburatani, Susumu Nishimura, Tomas LindahlAbstract:Abstract 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 andEscherichia 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. Thek 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 NaBH4trapping 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.
Jacques Laval - One of the best experts on this subject based on the ideXlab platform.
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the type of DNA Glycosylase determines the base excision repair pathway in mammalian cells
Journal of Biological Chemistry, 1999Co-Authors: Paola Fortini, Olga M Sidorkina, Jacques Laval, Eleonora Parlanti, Eugenia DogliottiAbstract:Abstract The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA Glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA Glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N 6-ethenoadenine (eA) are both substrates for the monofunctional 3-methyladenine DNA Glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA Glycosylase/β-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, eA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and eA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase β-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA Glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.
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role of lysine 57 in the catalytic activities of escherichia coli formamidopyrimidine DNA Glycosylase fpg protein
Nucleic Acids Research, 1998Co-Authors: Olga Sidorkina, Jacques LavalAbstract:The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA Glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA Glycosylase activity and Schiff base formation. The DNA Glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA Glycosylase activity of the Fpg protein in vitro and in vivo.
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3 n4 ethenocytosine a highly mutagenic adduct is a primary substrate for escherichia coli double stranded uracil DNA Glycosylase and human mismatch specific thymine DNA Glycosylase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Murat Saparbaev, Jacques LavalAbstract:Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N4-ethenocytosine (ɛC), 1,N6-ethenoadenine (ɛA), N2,3-ethenoguanine, and 1,N2-ethenoguanine cause mutations. The ɛA residues are excised by the human and the Escherichia coli 3-methyladenine-DNA Glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing ɛC residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove ɛC residues by a DNA Glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA Glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA Glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and ɛC-DNA Glycosylase activity, the dsUDG protein repairs thymine in a G/T mismatch. The fact that ɛC is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.
Bertrand Castaing - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the lactococcus lactis formamidopyrimidine DNA Glycosylase bound to an abasic site analogue containing DNA
The EMBO Journal, 2002Co-Authors: Laurence Serre, Karine Pereira De Jesus, Serge Boiteux, Charles Zelwer, Bertrand CastaingAbstract:The formamidopyrimidine-DNA Glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA Glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60° bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.
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crystal structure of the lactococcus lactis formamidopyrimidine DNA Glycosylase bound to an abasic site analogue containing DNA
The EMBO Journal, 2002Co-Authors: Laurence Serre, Karine Pereira De Jesus, Serge Boiteux, Charles Zelwer, Bertrand CastaingAbstract:The formamidopyrimidine-DNA Glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA Glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.
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DNA containing a chemically reduced apurinic site is a high affinity ligand for the e coli formamidopyrimidine DNA Glycosylase
Nucleic Acids Research, 1992Co-Authors: Bertrand Castaing, Serge Boiteux, Charles ZelwerAbstract:Abstract The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA Glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA Glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.