Date of Award
Doctor of Philosophy (PhD)
It is crucial that DNA damage is repaired efficiently in order to maintain genome integrity. Double-strand breaks, or DSBs, are the most harmful type of DNA damage, and thus their repair is tightly regulated. Fission yeast is a prime model system for studies of such repair processes as they favor homology-directed repair (HDR) over other more error-prone mechanisms. My work seeks to better interrogate homology-directed repair of DSBs in a temporally and spatially controlled manner, expanding on previous studies using primarily genetic outcome-based assays of repair. This is necessary in order to probe the complex dynamics of interhomologue repair in living cells. I have developed a microscopy-based assay in live diploid fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. My data indicate a highly efficient homology search in this system. Surprisingly, I observe not one but multiple site-specific and Rad51-dependent co-localization events between the DSB and donor. This and other observations suggest that efficient interhomologue repair in fission yeast often involves multiple strand invasion events that are regulated by Rqh1. In the absence of Rqh1, successful repair requires a single strand invasion event, suggesting that multiple strand invasion cycles reflect ongoing synthesis-dependent strand annealing (SDSA). However, failure to repair is also more likely in Rqh1 null cells, which could reflect increased strand invasion at non-homologous sites. This has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM (the human ortholog of Rqh1) and characterized by aberrant sister chromatid crossovers. Additionally, I monitored DSB repair dynamics under a variety of perturbations such as loss of repair factors or manipulations of the donor sequence. I found that fission yeast HDR is largely robust to these changes; chromatin mobility is not necessarily tied to repair efficiency; and donor sequence alterations can greatly affect associations with the DSB during homology search. Lastly, I discuss the implications of multiple strand invasions in HDR processing and also call for further work to expand on my research. In particular, it would be powerful to include complementary assays to assess sequence changes and strand invasion intermediates upon DSB induction in my system.
Vines, Amanda Jane, "Live Cell Dynamics of Homology-Directed DNA Double-Strand Break Repair" (2021). Yale Graduate School of Arts and Sciences Dissertations. 121.