Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase

Sanjay Adhikari, Aykut Üren, Rabindra Roy

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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,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. 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.

Original languageEnglish (US)
Pages (from-to)1201-1206
Number of pages6
JournalDNA Repair
Volume8
Issue number10
DOIs
StatePublished - Oct 2 2009

Keywords

  • Base excision repair
  • Glycosylase
  • N-Methylpurine-DNA glycosylase (MPG)
  • Product inhibition

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase'. Together they form a unique fingerprint.

Cite this