Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase.
N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N(6)-ethenoadenine (varepsilonA) from varepsilonA-containing oligonucleotide (varepsilonA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (K(D)) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for varepsilonA-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or varepsilonA-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.
Adhikari
Sanjay
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States.
Uren
Aykut
A
Roy
Rabindra
R
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D006027:Glycosides / Q000737:chemistry; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D020349:Surface Plasmon Resonance
D006027:Glycosides; C004981:1,N(6)-ethenoadenine; D019271:Hypoxanthine; D004247:DNA; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II; D000225:Adenine