Lys98 Substitution in Human AP Endonuclease 1 Affects the Kinetic Mechanism of Enzyme Action in Base Excision and Nucleotide Incision Repair Pathways

Nadezhda A Timofeyeva,Vladimir V Koval,Alexander A Ishchenko,Murat K Saparbaev,Olga S Fedorova,Vladimir N Uversky

doi:10.1371/journal.pone.0024063

Abstract

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5′-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates.

Highlights

There are a number of DNA lesions that continuously arise in the human genome, resulting in cell death, tumors, autoimmune diseases and aging
We examined the changes in fluorescence intensity of a 2-aPu residue located in the substrate at the 59-side of DHU in the presence of enzyme in nucleotide incision repair (NIR) buffer, in order to determine the kinetic mechanism of APE1K98A interaction with DHU-sub
To incise the DNA sugar-phosphate backbone at 59 to such structurally unrelated lesions as AP site and DHU, it is necessary that apyrimidinic endonuclease 1 (APE1) should undergo the conformational change of its active site region

Summary

Introduction

There are a number of DNA lesions that continuously arise in the human genome, resulting in cell death, tumors, autoimmune diseases and aging. It was estimated that about 104 of DNA lesions were generated per mammalian cell per day [1]. The majority of DNA damage can arise either spontaneously or following exposure to reactive oxygen species, derived from a wide range of cellular processes. Cellular genome integrity is sustained by several distinct repair mechanisms that remove DNA damage. The majority of the damaged DNA bases are eliminated through the base excision repair (BER) pathway [2]. BER is initiated by DNA glycosylases, enzymes that excise damaged and/or mispaired bases to produce apurinic/apyrimidinic sites (AP sites). During BER DNA is hydrolytically nicked 59 to the AP site by apurinic/ apyrimidinic endonucleases (AP endonucleases). APE1 (35.5 kDa) is the major AP endonuclease in human cells [6,7]

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Conclusion