UC San Diego

Regulation of chromatin dynamics is crucial for DNA- mediated processes including transcription and DNA damage repair. The glucanosyltransferase Gas1, which has been elegantly studied at the cell wall, was recently found to influence transcriptional silencing. The role of Gas1 in silencing was dependent on the catalytic activity of Gas1 and was separable from its cell wall functions. My research has focused on further characterization of gas1[Delta] chromatin-associated phenotypes and genetic interactions as well as analysis of the role of Gas1 localization in relation to chromatin dynamics. Interestingly, the gas1[Delta] strain is broadly sensitive to DNA damaging agents. In the gas1[Delta] mutant, initial sensing of DNA damage is intact, as observed by H2A serine 129 phosphorylation levels following exposure to genotoxins. However, both Rad53 phosphorylation and the DNA damage replication checkpoint are impaired following exposure to select genotoxins. The DNA damage sensitivity of the gas1[Delta] mutant is separable from the cell wall functions of Gas1 but is dependent on its [Beta]-1,3- glucanosyltransferase activity. Analysis of gas1[Delta] genetic interactions with genes encoding the major histone H3 acetyltransferases revealed that whereas the gas1[Delta] gcn5[Delta] double mutant is synthetically lethal, deletion of SAS3 leads to select mutual suppression of silencing defects. These results suggest that Gas1 and Sas3 may act to balance the distribution of silencing factors. Further, deletion of SAS3 specifically suppresses gas1[Delta] sensitivity to genotoxins that trigger the DNA replication checkpoint, leading to restoration of Rad53 phosphorylation and the cell cycle checkpoint. These findings suggest that Sas3 is antagonistic to the DNA replication checkpoint, which is unique and opposite to the role of the functionally overlapping acetyltransferases Gcn5. Finally, mutational analysis of select Gas1 domains demonstrates that blocking GPI anchor attachment, and thus passage of Gas1 through the secretory pathway, still permits rescue of Gas1's chromatin-associated functions. Our findings thus suggest that there may be two pools, or isoforms, of Gas1 that are functionally distinct. Overall, my research identifies new functions for both Gas1 and Sas3 and expands our understanding of Gas1's chromatin functions. In addition, I have identified a separation-of-function mutant that provides insight into the mechanism of Gas1 influence on chromatin dynamics