title: Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival.
abstract: Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.
authors.affiliation: Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, WA 98195-7705, USA.
authors.forename: Cheng-Yao
authors.initials: CY
authors.lastname: Chen
authors.forename: Haiwei H
authors.initials: HH
authors.lastname: Guo
authors.forename: Dharini
authors.initials: D
authors.lastname: Shah
authors.forename: A
authors.initials: A
authors.lastname: Blank
authors.forename: Leona D
authors.initials: LD
authors.lastname: Samson
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D000596:Amino Acids / Q000378:metabolism*; D020134:Catalytic Domain; D018736:DNA Adducts / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects*; D005816:Genetic Complementation Test; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D050296:Microbial Viability / Q000187:drug effects*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D009429:Netropsin / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D000596:Amino Acids; D018736:DNA Adducts; D050505:Mutant Proteins; C110963:methyl lexitropsin; D009429:Netropsin; D008741:Methyl Methanesulfonate; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases