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

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.

SUBMITTER: Morris LP

PROVIDER: S-EPMC3572540 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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

Morris Lydia P LP Degtyareva Natalya N Sheppard Clayton C Heyburn Lanier L Ivanov Andrei A AA Kow Yoke Wah YW Doetsch Paul W PW

DNA repair 20120719 9

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 substitut ...[more]

PMID: 22818187

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Project description:Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a long-lived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPS-related transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased non-adaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional co-regulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasome-mediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and age-related disease in humans.

Dataset Information

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

Publications

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

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