Effects of substrate specificity on initiating the base excision repair of N-methylpurines by variant human 3-methyladenine DNA glycosylases.
The human 3-methyladenine (AAG, ANPG, MPG) DNA glycosylase excises alkylated purines from DNA. In previous studies, we determined the importance of an active site amino acid (asparagine 169) in the recognition of substrates by AAG. In this study, we characterize the consequences of expressing the AAG variants bearing amino acid substitutions at position 169 in Saccharomyces cerevisiae that lack endogenous 3-methyladenine DNA glycosylase. Survival, mutation induction, and DNA double strand break formation were determined in response to methyl methanesulfonate. The ability of purified wild-type and AAG variants to remove 3-methyladenine and 7-methylguanine, the two most abundant adducts produced by methyl methanesulfonate, was also determined. The N169D AAG variant displayed a approximately 100-fold lower activity for 3-methyladenine as compared to wild-type and did not detectably remove 7-methylguanine. When expressed in S. cerevisiae, the N169D variant provided better protection against methyl methanesulfonate toxicity than wild-type. Fewer strand breaks in vivo were also seen in the presence of the N169D variant following MMS exposure. In contrast, the N169A and N169S AAG variants displayed approximately 30-fold lower activity for 3-methyladenine and 7-methylguanine. Expression of the N169A and N169S AAG variants in S. cerevisiae during methyl methanesulfonate exposure resulted in greater sensitivity, greater mutation induction following MMS exposure, and more strand breaks in vivo. Strand breaks seen in S. cerevisiae that express wild-type AAG or the N169 variants were resolved to varying extents during recovery. In contrast, strand breaks formed in S. cerevisiae that expressed a catalytically inactive AAG variant were not resolved during the recovery times examined. Taken together, the results provide evidence that 3-methyladenine adducts not repaired by base excision repair cause double strand breaks that are not rapidly resolved. Evidence is also provided that the BER intermediates resulting from excision of 7-methylguanine by wild-type AAG contributes to the mutagenicity and cytotoxicity of alkylating agents.
Connor
Ellen E
EE
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina 29208, USA.
Wilson
Jacqueline J
JJ
Wyatt
Michael D
MD
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D005818:Genetic Engineering; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D009153:Mutagens; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D008741:Methyl Methanesulfonate; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II; D000225:Adenine