Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Aurore Gelin,Modesto Redrejo-Rodríguez,Murat Saparbaev,Jacques Laval,Alexander A Ishchenko,Olga S Fedorova,Martin G Marinus

doi:10.1371/journal.pone.0012241

Abstract

BackgroundHuman apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.Methodology/Principal FindingWe identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.Conclusion/SignificanceTaken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.

Highlights

Oxidative damage to DNA caused by reactive oxygen species is believed to be a major type of endogenous cellular damage, and more than 80 different oxidative modifications of DNA bases and sugar backbone have been identified to date [1]
We showed that the truncated apyrimidinic endonuclease 1 (APE1) (ND61-APE1) lacking the first 61 N-terminal amino acids exhibits reduced nucleotide incision repair (NIR) but normal AP endonuclease activities demonstrating the feasibility of mutational separation of various DNA repair activities in a human DNA repair enzyme [11]
It was shown that R177 interacts with the 39 phosphate of an AP site in the major groove through a hydrogen bond, and the R177A mutant exhibits an enhanced AP endonuclease activity compared to WT APE1 [34]

Summary

Introduction

Oxidative damage to DNA caused by reactive oxygen species is believed to be a major type of endogenous cellular damage, and more than 80 different oxidative modifications of DNA bases and sugar backbone have been identified to date [1]. Closely located multiple base and/or sugar modifications as well as strand breaks, forming so-called clustered lesions, can be refractory to excision by DNA glycosylases [12,13,14]. These sites of clustered DNA damage could be processed by AP endonucleases without generating 39blocking intermediates since AP endonucleases only cut damaged DNA without removing the lesions [15]. Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. Physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells

Methods

Results

Conclusion