Title: Cellular responses that preserve genome stability in Saccharomyces cerevisiae
Description: To ensure the faithful transmission of genetic material to each progeny cell, DNA replication has to be accomplished faithfully and the duplicated chromosomes have to be distributed equally to the daughter cells. My dissertation focuses on the MSA1 gene, which functions as a transcription factor to facilitate the DNA replication during S-phase, and HSK3 , which functions in the DASH complex to ensure the proper segregation of chromosomes in mitosis. DRC1 is isolated as a genomic suppressor of dpb11-1 and forms the initiator complex with Dpb11 that facilitates the recruitment of DNA polymerase to origins. The drc1-1 mutant shows sensitivity to the replication inhibitor, hydroxyurea. In the first study, we identify a cell cycle regulated transcription factor, MSA1 , as a suppressor of drc1-1 . MSA1 overproduction also suppresses the temperature sensitivity of dpb11-1 and pol2-12 (the catalytic subunit of DNA polymerase [varepsilon]). Conversely, msa1 deletion exacerbates the mutant phenotypes of both drc1-1 and dpb11-1 and msa1 deletion alone results in a delay in S phase entry, which suggests a positive role for MSA1 in DNA replication. MSA1 represents a new cell cycle regulated gene important for S phase entry.

Ask1 is a subunit of the DASH complex, which mediates the interaction between kinetochores and spindles. The DASH complex is required for spindle integrity and bipolar attachment of sister chromatids to the mitotic spindles. In the second study, we isolate a novel gene and components of the Ras/Protein Kinase A pathway as suppressors of ask1-2 and ask1-3 mutants. The novel gene, HSK3 ( H elper of As k 1), is an essential protein of 69 amino acids. Hsk3 shares characteristics with the DASH complex, including: the localization at spindles and the association with centromeric DNA in a spindle-dependent manner. We demonstrate that Hsk3 is part of the DASH complex and it plays a critical role in maintaining the integrity of the DASH complex and propose that Hsk3 acts to incorporate Ask1 into the DASH complex. In addition, we show that over-expression of PDE2 (phosphodiesterase that removes cAMP to shut-off PKA activation) or deletion of RAS2 rescues the temperature sensitivity of ask1-3 mutants. We propose that Ras2 negatively regulates DASH through the PKA pathway. Overall our results facilitate the understanding of these two key events of yeast cells by demonstrating: (1) MSA1 functions during S-phase to facilitate completion of DNA replication in a timely fashion; (2) HSK3 serves to preserve the integrity of the DASH complex required for the subsequent proper segregation of chromosomes.