Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

Saccharomyces cerevisiae Apn1 mutation affecting stable protein expression mimics catalytic activity impairment: implications for assessing DNA repair capacity in humans.

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.