EXCISED DAMAGED BASE DETERMINES THE TURNOVER OF HUMAN N-METHYLPURINE-DNA GLYCOSYLASE

摘要

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’s enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1, N⁶-ethenoadenine (εA) from εA-containing oligonucleotide (εA-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 (KD) 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 εA-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 εA-DNA reaction is. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG’s glycosylase actiity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.