Issue 5, 2016

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

Abstract

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.

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

Article information

Article type
Paper
Submitted
18 Feb 2016
Accepted
04 Apr 2016
First published
06 Apr 2016
This article is Open Access
Creative Commons BY license

Mol. BioSyst., 2016,12, 1527-1539

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

A. D. Miroshnikova, A. A. Kuznetsova, Y. N. Vorobjev, N. A. Kuznetsov and O. S. Fedorova, Mol. BioSyst., 2016, 12, 1527 DOI: 10.1039/C6MB00128A

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