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UBC Theses and Dissertations

Diverse functions of Rtt107 in the Saccharomyces cerevisiae DNA damage response Brown, Joshua Allen Richard


Cells are routinely confronted with DNA damage and depend on genome maintenance factors to prevent cell death, as well as mutations and genomic rearrangements. The DNA damage response includes DNA repair and cell cycle checkpoints, which arrest sensitive processes such as DNA replication while damage is repaired. Rtt107 is an evolutionarily conserved Saccharomyces cerevisiae protein that is required for resistance to agents that cause replicative stress and DNA damage. Rtt107 has been best characterized as a scaffold that acts in response to DNA damage, localizing to phosphorylated H2A and binding to diverse protein partners, including Slx4. However, other functions of Rtt107 remain unclear. Mutants lacking Rtt107 exhibit prolonged checkpoint activation after replicative stress, and the “checkpoint dampening” model proposes that Rtt107-Slx4 reduces checkpoint activity by limiting formation of a Rad9-Dpb11 complex that promotes checkpoint activity. In this dissertation, I identified higher levels of DNA damage in cells lacking Rtt107 during replicative stress. This presented an alternative cause of checkpoint activity, and I showed that the Rad9-Dpb11 complex was not primarily responsible for prolonged checkpoint activity in this context. Based on these findings, I proposed a revised model of Rtt107 function during replicative stress, in which Rtt107 primarily reduced checkpoint activity by limiting DNA damage levels. I next explored Rtt107’s less well-understood function in limiting spontaneous genome instability, and found that this was partially distinct from its function in the context of treatment with replicative stress. Specifically, Rtt107 bound to the DNA repair protein Rad55 to limit spontaneous loss of heterozygosity, and spontaneous crossover events. I next focused on the regulation of Rtt107, specifically the phosphorylation of Rtt107’s SQ/TQ cluster domain (SCD) by the Mec1 kinase after treatment with DNA damaging agents. To address inconsistencies between prior reports on the importance of Rtt107 phosphorylation, I reviewed prior work on this subject and demonstrated that phosphorylation of Rtt107’s SCD was dispensable for growth during chronic treatment with DNA damaging agents. Collectively, my dissertation reveals how Rtt107 acts with various partners and in different contexts to confer resistance to agents that cause replicative stress and DNA damage, and to prevent spontaneous genome instability.

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