Dissecting the Broad Substrate Specificity of Human 3-Methyladenine-DNA Glycosylase

Patrick J O'Brien,Tom Ellenberger

doi:10.1074/jbc.m312232200

Abstract

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.

Highlights

DNA bases undergo spontaneous deamination and can be alkylated by reactive cellular metabolites and environmental mutagens [1]
Base flipping contributes to specificity as destabilized mismatched base pairs are better substrates than stable WatsonCrick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by recognizing each lesion
The rate enhancement, which is defined as kcat/knon, provides a measure of how well the active site environment stabilizes the transition state relative to that of the nonenzymatic reaction, and it does not include all of the entropic effects of bringing substrates together at the active site

Summary

EXPERIMENTAL PROCEDURES

DNA substrates of the sequence 5Ј-CGATAGCATCCTXCCTTCTCTCCAT annealed to the complementary oligonucleotide 5Ј-ATGGAGAGAAGGYAGGATGCTATCG, in which lesion X is paired with base Y (X1⁄7Y), were prepared as described previously [12]. The catalytic domain of human AAG (⌬80) in which the N-terminal 80 amino acids have been deleted was overexpressed in Escherichia coli and purified [12] This domain has glycosylase activity identical to the full-length protein [4, 12]. To ensure single turnover conditions, the concentration of AAG was kept in excess of the concentration of labeled DNA (ϳ1 nM). Control reactions in which single-stranded DNA was incubated under these reaction conditions revealed 10 –30-fold greater rates of hydrolysis (data not shown). This observation is consistent with a previous study that utilized genomic DNA [14], and it suggests that these short oligonucleotide duplexes were stable under our experimental conditions. A negative value of ⌬⌬G‡ indicates that a given substituent contributes to catalytic recognition by AAG (Reactions 2– 8)

RESULTS

30 Ϯ 10a 31 Ϯ 6a

DISCUSSION