Saccharomyces cerevisiae Apn1 Mutation Affecting Stable Protein Expression Mimics Catalytic Activity Impairment: Implications for Assessing DNA Repair Capacity in Humans

• Type of Material: Article
• Authors: Lydia P. Morris, Emory University; Natalya Degtyareva, Emory University; Clayton Sheppard, Emory University; Lanier Heyburn, Emory University; Andrey Andreyevich Ivanov, Emory University; Yoke Wah Kow, Emory University; Paul Doetsch, Emory University
• Language: English
• Date: 2012-09-01
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 Elsevier B.V. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1016/j.dnarep.2012.06.008
• Title of Journal or Parent Work: DNA Repair
• ISSN: 1568-7864
• Volume: 11
• Issue: 9
• Start Page: 753
• End Page: 765
• Grant/Funding Information: This work was supported by NIEHS grant ES011163.
• Keywords: Methyl methanesulfonate; AP endonuclease; Single nucleotide polymorphism; Apn1; Structure-function relationship
• Research Categories: Chemistry, Biochemistry; Health Sciences, Oncology

Abstract

Apurinic/apyrimidinic (AP) endonucleases play a major role in the repair of AP sites, oxidative damage and alkylation damage in DNA. We employed Saccharomyces cerevisiae in an unbiased forward genetic screen to identify amino acid substitutions in the major yeast AP endonuclease, Apn1, that impair cellular DNA repair capacity by conferring sensitivity to the DNA alkylating agent methyl methanesulfonate. We report here the identification and characterization of the Apn1 V156E amino acid substitution mutant through biochemical and functional analysis. We found that steady-state levels of Apn1 V156E were substantially decreased compared to wild type protein, and that this decrease was due to more rapid degradation of mutant protein compared to wild type. Based on homology to E. coli endonuclease IV and computational modeling, we predicted that V156E impairs catalytic ability. However, overexpression of mutant protein restored DNA repair activity in vitro and in vivo. Thus, the V156E substitution decreases DNA repair capacity by an unanticipated mechanism via increased degradation of mutant protein, leading to substantially reduced cellular levels. Our study provides evidence that the V156 residue plays a critical role in Apn1 structural integrity, but is not involved in catalytic activity. These results have important implications for elucidating structure-function relationships for the endonuclease IV family of proteins, and for employing simple eukaryotic model systems to understand how structural defects in the major human AP endonuclease APE1 may contribute to disease etiology.