Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion

The human alkyladenine DNA glycosylase has a broad substrate specificity, e xcising a structurally diverse group of damaged purines from DNA. To more c learly define the structural and mechanistic bases for substrate specificit y of human alkyladenine DNA glycosylase, kinetics of excision and DNA bindi ng activities were measured for several different damaged and undamaged pur ines within identical DNA sequence contexts. We found that 1,N-6-ethenoaden ine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, w hereas 7,8-dihydro-8-oxoguanine, O-6-methylguanine, adenine, and guanine we re 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 whe reas binding assays showed that the binding affinity was about five times g reater for epsilonA than for fix, The opposing pyrimidine base had a signif icant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for EA. Surprisingly, replacing a T with a U oppos ite Hx dramatically reduced the excision rate by a factor of 15 and increas ed the affinity by a factor of 7-8, The binding affinity of human alkyladen ine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.