Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase

Sanjay Adhikari, Jeffery A. Toretsky, Linshan Yuan, Rabindra Roy

Research output: Contribution to journalArticle

43 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. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase β, and ligases, require Mg2+. Here we have demonstrated that Mg2+ can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg2+ at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg2+ affected K m, but not Vmax, and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg2+ stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg2+ inhibits MPG activity by abrogating substrate binding. Because Mg2+ is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.

Original languageEnglish (US)
Pages (from-to)29525-29532
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number40
DOIs
StatePublished - Oct 6 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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