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J. Biol. Chem., Vol. 278, Issue 20, 18289-18296, May 16, 2003

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The Exonuclease Activity of Human Apurinic/Apyrimidinic Endonuclease (APE1)
BIOCHEMICAL PROPERTIES AND INHIBITION BY THE NATURAL DINUCLEOTIDE Gp₄G^*

Kai-ming Chou and Yung-chi Cheng

From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520

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 K_m value for matched, 3' mispaired, or nucleoside analog -L-dioxolane-cytidine terminated nicked DNA as well as for DNA containing a tetrahydrofuran, an abasic site analog. The k_cat for exonuclease activity on matched, 3' mispaired, and -L-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min¹, respectively, and 787.5 min¹ 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-, deoxyribo-nucleoside/nucleotides, NAD⁺, NADP⁺, and Ap₄A. The K_i values of Gp₄G for the endonuclease and exonuclease activities of APE1 are 10 ± 0.6 and 1 ± 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.

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