Abstract
The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N(6)-ethenoadenine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O(6)-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and epsilonA paired with T showed that excision of Hx was about four times faster than epsilonA, whereas binding assays showed that the binding affinity was about five times greater for epsilonA than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for epsilonA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.
Highlights
The base excision repair pathway provides the cell with a major line of defense against damage to DNA bases by excising damaged bases and resynthesizing DNA
B, the fraction of ⑀A excised during the electrophoretic mobility shift assays (EMSAs) time course was quantitated and is plotted along with the fraction of ⑀A1⁄7T DNA bound by human alkyladenine DNA glycosylase (hAAG)⌬79 and hAAG⌬79E125Q
Various studies have shown that hAAG is capable of excising 3-methyladenine [5,6,7,8,9,10], 7-methylguanine [5,6,7, 10, 11], 1,N6-ethenoadenine [8, 9, 11, 12], etheno adducts of guanine [12], 7,8-dihydro-8-oxoguanine [13], hypoxanthine [11, 14, 15], and undamaged purines [16, 17]
Summary
Varying the base paired with a damaged DNA base were examined. The alkylated DNAs processed by hAAG appear to have few characteristics in common, the goal of our experiments is to determine whether there are common underlying structural features that are recognized by hAAG. Base excision and DNA binding by hAAG were measured for more than 20 different base pair combinations. We found ethenoadenine (⑀A) and hypoxanthine (Hx) to be the most efficiently excised; excision of Hx was affected dramatically by its base-pairing partner
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