Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair.
Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.
Horton
J K
JK
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Prasad
R
R
Hou
E
E
Wilson
S H
SH
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002457:Cell Extracts / Q000494:pharmacology; D018736:DNA Adducts / Q000378:metabolism; D019175:DNA Methylation; D019951:DNA Polymerase beta / Q000235:genetics / Q000494:pharmacology*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005347:Fibroblasts / Q000187:drug effects*; D017353:Gene Deletion; D006898:Hydroxylamines / Q000494:pharmacology; D008190:Lyases / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D051379:Mice; D008957:Models, Genetic; D009153:Mutagens / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays; D014498:Uracil / Q000378:metabolism
D000477:Alkylating Agents; D002457:Cell Extracts; D018736:DNA Adducts; D006898:Hydroxylamines; D009153:Mutagens; D012267:Ribosemonophosphates; D014498:Uracil; D008770:Methylnitrosourea; C046346:5-deoxyribose 1-phosphate; C005214:methoxyamine; D008741:Methyl Methanesulfonate; D019951:DNA Polymerase beta; D008190:Lyases