Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1

Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K+) and divalent (Mg2+, Mn2+, Ca2+, Zn2+, Cu2+, and Ni2+) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K+ ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu2+ ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca2+ ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn2+ < Ni2+ < Mn2+ < Mg2+. Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg2+ ions stabilize the protein structure and the enzyme-substrate complex.