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, 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.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
C W
CW
Abner
A Y
AY
Lau
T
T
Ellenberger
L B
LB
Bloom
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D006801:Humans; D006860:Hydrogen Bonding; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D019271:Hypoxanthine; C007640:1,N(6)-ethenoadenosine; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000241:Adenosine