Mammalian DNA beta-polymerase in base excision repair of alkylation damage.
DNA beta-polymerase (beta-pol) carries out two critical enzymatic reactions in mammalian single-nucleotide base excision repair (BER): DNA synthesis to fill the repair patch and lyase removal of the 5'-deoxyribose phosphate (dRP) group following cleavage of the abasic site by apurinic/apyrimidinic (AP) endonuclease (1). The requirement for beta-pol in single-nucleotide BER is exemplified in mouse fibroblasts with a null mutation in the beta-pol gene. These cells are hypersensitive to monofunctional DNA methylating agents such as methyl methane-sulfonate (MMS) (2). This hypersensitivity is associated with an abundance of chromosomal damage and induction of apoptosis and necrotic cell death (3). We have found that beta-pol null cells are defective in repair of MMS-induced DNA lesions, consistent with a cellular BER deficiency as a causative agent in the observed hypersensitivity. Further, the N-terminal 8-kDa domain of beta-pol, which contains the dRP lyase activity in the wild-type enzyme, is sufficient to reverse the methylating agent hypersensitivity in beta-pol null cells. These results indicate that lyase removal of the dRP group is a pivotal step in BER in vivo. Finally, we examined MMS-induced genomic DNA mutagenesis in two isogenic mouse cell lines designed for study of the role of BER. MMS exposure strongly increases mutant frequency in beta-pol null cells, but not in wild-type cells. With MMS treatment, beta-pol null cells have a higher frequency of all six base-pair substitutions, suggesting that BER plays a role in protecting the cell against methylation-induced mutations.
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Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Wilson
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D000478:Alkylation; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D010582:Bacteriophage lambda / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000502:physiology; D004249:DNA Damage; D011088:DNA Ligases / Q000502:physiology*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005838:Genotype; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008745:Methylation; D051379:Mice; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity; D019761:Phosphorus-Oxygen Lyases / Q000502:physiology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012267:Ribosemonophosphates / Q000737:chemistry
D009153:Mutagens; D011993:Recombinant Fusion Proteins; D012267:Ribosemonophosphates; D001224:Aspartic Acid; C046346:5-deoxyribose 1-phosphate; D008741:Methyl Methanesulfonate; C423906:5'-deoxyribose phosphate lyase; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D019761:Phosphorus-Oxygen Lyases; D011088:DNA Ligases