Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2008-9-22
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1568-7864
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
7
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1731-45
pubmed:meshHeading
pubmed-meshheading:18706524-Amino Acid Sequence, pubmed-meshheading:18706524-Amino Acid Substitution, pubmed-meshheading:18706524-Amino Acids, pubmed-meshheading:18706524-Catalytic Domain, pubmed-meshheading:18706524-DNA Adducts, pubmed-meshheading:18706524-DNA Glycosylases, pubmed-meshheading:18706524-Escherichia coli, pubmed-meshheading:18706524-Genetic Complementation Test, pubmed-meshheading:18706524-Humans, pubmed-meshheading:18706524-Methyl Methanesulfonate, pubmed-meshheading:18706524-Microbial Viability, pubmed-meshheading:18706524-Molecular Sequence Data, pubmed-meshheading:18706524-Mutagenesis, pubmed-meshheading:18706524-Mutant Proteins, pubmed-meshheading:18706524-Mutation, pubmed-meshheading:18706524-Netropsin, pubmed-meshheading:18706524-Protein Binding, pubmed-meshheading:18706524-Protein Structure, Secondary, pubmed-meshheading:18706524-Structure-Activity Relationship, pubmed-meshheading:18706524-Substrate Specificity
pubmed:year
2008
pubmed:articleTitle
Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival.
pubmed:affiliation
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, WA 98195-7705, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural