Repair of AP Sites and Aging in Saccharomyces cerevisiae

Universidad Central Del Caribe, Bayamon PR

Investigators

Linked publications, trials & patents

Abstract

The long-term goal of this study is to determine the role of DNA damage, specifically apurinic/apyrimidinic (AP or abasic) sites, in the aging process. AP sites are formed by spontaneous loss of bases and during base excision repair. AP sites are one of the most common DNA lesions present in DNA and their accumulations have been associated to aging. The Saccharomyces cerevisiae SGSI is the homolog of the Werner's Syndrome gene, a premature aging syndrome and Bloom's syndrome, a cancer-prone syndrome. SGSI encodes a DNA helicase with proposed roles in DNA replication, DNA replication, repair, recombination, and transcription. Yeast strains that lack the SGS1 gene are sensitive to methyl methasulfonate (MMS) an agent that generates AP sites suggesting that SGS1 may be required during DNA repair processes. SGS1 may be involved in DNA repair processes in at least two possible ways. First, SGS1 may participate in the repair of AP sites via base excision repair. Second, SGS1 may be required for the tolerance of DNA damage, a mechanism known as post-replicational repair. In this grant proposal we will test the hypothesis that SGS1 is required for the repair and/or tolerance of AP sites and that accumulation of AP sites via base excision repair we will perm an epistasis analysis of the sgs1delta mutation with respect to APN1 and APN2 genes, which are involved in the repair of AP sites. We will also determine if sgs1delta yeast strains are deficient in the repair of AP sites by means of alkaline sucrose sedimentation analysis of uniformly labeled DNA after MMS treatment. To determine if SGS1 is involved in post-replicational repair of AP sites we will perform an epistasis of the sgs1 delta mutation with respect to genes that belong to the RAD6 epistatic group. We will also perform an in vivo post-replicational repair assay that monitors the conversion of low molecular weight DNA into high molecular weight DNA in the newly synthesized DNA strand after DNA damage. Finally, we will study the contribution of AP site accumulation in yeast aging by determining, in strains that are deficient in the repair of AP sites, the following parameters: life-span, loss of transcriptional silencing, and generation of rDNA extrachromosomal circles. Completion of these specific aims may establish a role for SGS1 in DNA repair and may define the role of a specific DNA lesion, the AP site in yeast aging.