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 (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) 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 Cu(2+) 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 Ca(2+) 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 Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex.

Molecular snapshots of APE1 proofreading mismatches and removing DNA damage

“…A C/T mismatch was chosen because observations in our lab, as well as data published by others, indicate it as an optimal substrate for APE1 exo activity 14 – 17 . Initial substrate complex crystals were generated in a solution containing CaCl 2 , which inhibits catalysis 18 , 19 , and resulted in clear electron density for the phosphate and sugar moiety. However, relatively poor density was observed for the base of the mismatched C. To confirm we obtained an uncut substrate complex, we used otherwise identical DNA with a phosphorothioate (PS) modification 5′ to the mismatched C. This modification substitutes sulfur for a non-bridging oxygen, preventing incision 11 , 20 , 21 .…”

The Role of Active-Site Plasticity in Damaged-Nucleotide Recognition by Human Apurinic/Apyrimidinic Endonuclease APE1

“…The DHU-substrate containing the smallest damaged pyrimidine base is cleaved with greater efficiency than αA and εA; therefore, this substrate was chosen for further detailed analysis of the substrate-binding process. It should be mentioned that the activity of APE1 depends on the concentration of Mg 2+ [ 14 , 34 ]. Typically, the AP-site cleavage activity is tested in the presence of ≥5 mM Mg 2+ [ 14 , 35 , 36 , 37 , 38 ], but the cleavage of DNA bearing a damaged base is tested at a low concentration of Mg 2+ , within the 0.01–0.5 mM range [ 12 , 14 , 35 , 36 , 39 ].…”

“…The reaction solution was supplemented with a certain concentration of MgCl 2 when required. Of note, our previous data [ 34 ] have revealed that supplementation of the solution of apo-APE1 and 1.0 mM EDTA by MgCl 2 (even at 0.05 mM) restores catalytic activity to some extent with subsequent saturation of the enzyme by the Mg 2+ ion with the increasing concentration of MgCl 2 . Therefore, in the present report, we used the same design of experiments.…”

Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII

“…The kinetic scheme contained n reversible and m irreversible steps, and an enzyme–product complex equilibrium step and corresponding kinetic parameters were derived essentially as described previously [7,29]. …”