Replication fork collapse and recombination-dependent replication restart at replication fork barriers

Thesis

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.

Authors: Tamang, S

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford