The exonuclease activity of human apurinic/apyrimidinic endonuclease (APE1). Biochemical properties and inhibition by the natural dinucleotide Gp4G.
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 beta-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 beta-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. Gp(4)G 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 Ap(4)A. The K(i) values of Gp(4)G for the endonuclease and exonuclease activities of APE1 are 10 +/- 0.6 and 1 +/- 0.2 microm, respectively. Given the relative concentrations of Gp(4)G, 3' mispaired, and abasic DNA, Gp(4)G 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 MgCl(2) or salt concentrations.
Chou
Kai-Ming
KM
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Cheng
Yung-Chi
YC
D000596:Amino Acids / Q000737:chemistry; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004148:Dioxolanes / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D000596:Amino Acids; D004148:Dioxolanes; D011994:Recombinant Proteins; D015636:Magnesium Chloride; C074908:troxacitabine; D009532:Nickel; D003596:Cytosine; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase