Rapid dissociation of human apurinic endonuclease (Ape1) from incised DNA induced by magnesium.
Repair of apurinic/apyrimidinic (AP) sites is initiated by AP endonucleases, such as the human Ape1 protein (also called Hap1, Apex, and Ref1). This and related enzymes show strong dependence on divalent cations, particularly magnesium. Here we explore the role of this metal in different stages of the Ape1 reaction: substrate binding, cleavage, and product release. We examined DNA binding using an electrophoretic approach and DNA cleavage in single-turnover and steady-state reactions. Magnesium at low to moderate concentrations accelerated both substrate and product release by wild-type Ape1 protein. For a mutant Ape1 protein with an aspartate to alanine substitution at residue 308, substrate in preformed protein-DNA complexes was more efficiently cleaved before release in contrast to wild-type Ape1, whereas product release was accelerated dramatically. The magnesium dependence of steady-state AP endonuclease reactions was sigmoidal for both wild-type and the aspartate 308 to alanine protein but was not sigmoidal for an aspartate 283 to alanine derivative of Ape1. These results show that magnesium affects both DNA interactions with and phosphodiester cleavage by Ape1 and can change the rate-limiting step of the reaction. Structural studies will need to be interpreted in the context of these diverse effects of the metal.
Masuda
Y
Y
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Bennett
R A
RA
Demple
B
B
D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects
D015636:Magnesium Chloride; D011189:Potassium Chloride; D004247:DNA; D004720:Endonucleases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium