The exonuclease activity of human apurinic/apyrimidinic endonuclease (APE1). Biochemical properties and inhibition by the natural dinucleotide Gp4G

Kai Ming Chou, Yung Chi Cheng

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

Human DNA apurinic/apyrimidinic endonuclease (APE1) plays a key role in the DNA base excision repair process. In this study, we further characterized the exonuclease activity of APE1. The magnesium requirement and pH dependence of the exonuclease and endonuclease activities of APE1 are significantly different. APE1 showed a similar Km value for matched, 3′ mispaired, or nucleoside analog β-L-dioxolane-cytidine terminated nicked DNA as well as for DNA containing a tetrahydrofuran, an abasic site analog. The kcat for exonuclease activity on matched, 3′ mispaired, and β-L-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min-1, respectively, and 787.5 min-1 for APE1 endonuclease. Site-directed APE1 mutant proteins (E96A, E96Q, D210E, D210N, and H309N), which target amino acid residues in the endonuclease active site, also showed significant decrease in exonuclease activity. Gp4G was the only potent inhibitor to compete against the substrates of endonuclease and exonuclease activities among all tested naturally occurring ribo-, deoxyribonucleoside/nucleotides, NAD+, NADP+, and Ap4A. The Ki values of Gp4G for the endonuclease and exonuclease activities of APE1 are 10 ± 0.6 and I ± 0.2 μM, respectively. Given the relative concentrations of Gp4G, 3′ mispaired, and abasic DNA, Gp4G may play an important role in regulating APE1 activity in cells. The data presented here suggest that the APE1 exonuclease and AP endonuclease are two distinct activities. APE1 may exist in two different conformations, and each conformation has a preference for a substrate. The different conformations can be affected by MgCl2 or salt concentrations.

Original languageEnglish (US)
Pages (from-to)18289-18296
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number20
DOIs
StatePublished - May 16 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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