Investigating the molecular mechanisms of break-induced replication in S. pombe

Thesis

Complete and accurate genome replication at each cell division is essential to the viability of all organisms. However, numerous obstacles can pose a barrier to DNA replication. In mammalian cells, single-strand DNA breaks (SSBs) are ubiquitous replication fork barriers (RFBs); it is estimated that over 10,000 SSBs occur in each mammalian cell every day, potentially arising from diverse endogenous and exogenous sources. When replisomes collide with SSBs, they may undergo collapse and fork breakage to generate single-ended double-strand DNA breaks (seDSBs). seDSBs may be channelled into repair by Break-Induced Replication (BIR); in contrast to alternative homologous recombination (HR) pathways, BIR is a highly mutagenic process, and BIR-associated genome instability has been implicated in numerous human disease contexts, including cancer. Understanding the molecular mechanisms driving and regulating BIR, and its tendency for genome destabilisation, is thus of significant clinical interest.

In this study, I employ a previously established template switch-based genetic assay – in conjunction with the ‘Flp-nick’ system for site-specific SSB generation – to examine the genetic requirements of SSB-induced BIR in S. pombe. I demonstrate that the Pfh1 helicase (previously implicated in both S. cerevisiae and mammalian BIR) is required for Flp-nick-induced template switching, providing robust evidence for the stimulation of bona fide BIR. I show that such Flp-nick-induced BIR in S. pombe is completely dependent upon Rad51, and that Rad52 strand annealing activity is also a key contributor to BIR-associated template switching. Further, I establish evidence that the non-homologous end joining protein Ku70 suppresses the induction of BIR. I also investigate the contribution of the short-range DNA resection enzymes Rad50 and Ctp1, and the F-box protein Pof3, to BIR.

In parallel to my template switch-focused experimental approach, I additionally establish a fluorescence live-cell imaging system to examine the extent of Rad52 recruitment to sites of recombination-inducing Flp-nicks. In contrast to previous observations for RTS1-induced replication perturbation, I do not detect substantial Flp-nick-induced recruitment of Rad52 foci. I also explore the use of a nuclear localisation signal (NLS) as a strategy for optimising our current Flp-nick system; despite driving a detectable increase in Flp protein nuclear localisation, an N-terminal NLS does not increase the level of detectable Flp-nick-induced, BIR-associated template switching. Finally, I examine the potential for replication slippage to contribute to BIR-associated genome destabilisation; utilising a reporter allele previously established as a substrate for replication slippage, I do not find evidence for replication slippage in the context of Cas9 nickase-induced BIR.

Authors: Holt, OM

• DOI: 10.5287/ora-2a4qpkmvb
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English