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Title: Replication fork collapse and recombination-dependent replication restart at replication fork barriers
Author: Tamang, Sanjeeta
ISNI:       0000 0004 7966 3463
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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In order for the genome of any organism to be successfully replicated its replication machinery has to overcome barriers in the template DNA. These so-called replication fork barriers (RFBs) can sometimes cause the replication fork to collapse. Replication fork collapse is a poorly understood process that is thought to involve either replisome remodelling and/or disassembly paving the way for homologous recombination (HR) proteins to act and restart replication, by a process known as recombination dependent replication restart (RDR), which is essential for completing DNA synthesis. In this thesis I present my work identifying factors that are instrumental in the replication restart process, with a particular focus on those factors that are needed to aid the transition from collapsed fork to one that is receptive to HR proteins. I have used the RTS1 site-specific RFB in fission yeast for this study as it acts as a potent polar RFB that triggers fork collapse and recombination protein recruitment. By screening candidate factors, I identified the PCNA unloader Elg1 as being strongly required for RTS1-induced recombination. By genetic experiments and live cell imaging, I show that unloading of PCNA by Elg1 promotes RDR by allowing efficient recruitment of Rad52 to the blocked replication fork and by limiting the anti-recombinogenic activity of Fbh1. I also provide evidence that Ctf18 acts in opposition to Elg1 to limit RTS1-induced recombination. Finally, I investigate the importance of Rad51, the central HR protein, and its mediators (Rad55-Rad57, Swi5-Sfr1, and Rdl1-Rlp1-Sws1) in RDR. By genetic analysis of RTS1-induced recombination, I discover that neither Rad51 nor its aforementioned mediators are essential for RDR. These findings indicate the existence of a robust Rad51-independent pathway of RDR in fission yeast.
Supervisor: Whitby, Matthew C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available