Elucidating Mechanisms Of Base Excision Repair And Genetic Instability In Saccharomyces Cerevisiae

A large subset of DNA damage acquired by cells is repaired by the base excision repair (BER) pathway. Though defects in many BER genes have been associated with neurodegenerative diseases and cancer, the molecular basis for such associations is not well understood. Further, when cells cannot repair oxidative DNA lesions normally targeted by BER, large-scale genome destabilization can occur. The major goals of the studies presented here are to better understand BER mechanisms at the level of individual proteins and on the genome-wide level. We employed Saccharomyces cerevisiae because the biochemical steps of BER are highly conserved, and S. cerevisiae is a well developed model for DNA repair studies. AP endonucleases play a central role in the repair of DNA damage through the BER pathway, thus our studies focus on the major yeast AP endonuclease, Apn1, to better understand how BER protects cells against genomic instability, an important characteristic of cancer. In an unbiased, forward genetic screen to identify mutations in APN1 that impair cellular DNA repair capacity we identified and characterized variant Apn1 V156E, which was predicted to decrease catalytic function based on homology modeling. We found that, unlike wild type Apn1, the V156E is targeted for degradation by a proteasome-independent mechanism, leading to decreased steady-state levels. Inducing transcription of APN1-V156E using a regulatable promoter restored protein to levels comparable to wild type Apn1 and functionally restored DNA repair capacity. Thus, the V156 residue plays a critical role in maintaining Apn1 protein levels and normal levels of repair independent of catalytic function. In genome-wide chromatin immunoprecipitation studies aimed at exploring the relationship between DNA damage repair and genomic instability using Apn1 as the target protein, we found that the level of oxidative stress dictates the distribution of Apn1 across the genome. Regardless of oxidative stress level, Apn1 binding sites are enriched for C and G nucleotides, suggesting that Apn1 targets particular regions in a base content-specific manner. These results have implications for understanding how the genomic distribution of DNA repair activities preserves genome integrity and for understanding how defects in the major human AP endonuclease may contribute to disease.