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Functional characterization of Rtt107 in the DNA damage response in Saccharomyces cerevisiae

University of British Columbia

Living cells are constantly exposed to DNA damage that if not properly repaired may lead to mutations or cell death. Due to the crucial importance of genome integrity, cells have complex response mechanisms to sense and repair DNA lesions. In my dissertation, I focus on a protein called Rtt107 in the DNA damage response in Saccharomyces cerevisiae. Yeast lacking Rtt107 are hypersensitive to DNA damaging agents, but the exact role of Rtt107 remains unclear. The DNA damage response occurs in the context of chromatin, and thus is affected by chromatin modifiers. I explored the connection between Rtt107 and the histone methyltransferase Dot1. Deletion of DOT1 or loss of its target H3 K79 trimethylation suppressed the DNA damage sensitivity of rtt107∆ mutants. This suppression was mediated by the translesion synthesis bypass pathway, which improved DNA damage resistance, but increased mutation rates. Rtt107 interacts physically with a number of DNA repair proteins, and I characterized its interaction with the SMC5/6 complex, which has numerous roles in chromosome maintenance. Rtt107 was required for the recruitment of SMC5/6 to double-stranded breaks, but not protein-bound nicks. However, these proteins also contributed independently to other genome maintenance functions. I mapped conditional genetic interaction networks to further understand the functions of Rtt107, its interaction partner Slx4, and a poorly characterized helicase, Hrq1. RTT107 had the greatest number of conditional genetic interactions, particularly after exposure to camptothecin, suggesting that it has an important role in responding to this type of lesion. The genetic interaction profiles and associated gene ontology terms of HRQ1 was the most similar to that of SLX4, suggesting that they may work together in the DNA damage response. Focusing more closely on the protein structure of Rtt107, I discovered that the C-terminal BRCT (BRCA1 C-terminal) domains bound to phosphorylated H2A, mediating the recruitment of Rtt107 to sites of DNA lesions. Rtt107 acted as a scaffold to recruit other proteins to these sites, but its requirement differed depending on the type of DNA insult. In summary, my dissertation contributes to the understanding of Rtt107 function, while highlighting key principles in the DNA damage response.

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2014-07-29

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/48514

Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/