Investigating break induced replication and template switching in fission yeast

Single-stranded DNA breaks (SSBs) are the most common DNA lesion a cell can suffer with ~1 SSB formed every 5 seconds in a mammalian cell under normal conditions. Hence, a significant number of SSBs are encountered by replication forks in S-phase and these encounters can result in double-stranded DNA breaks (DSBs), which are highly toxic lesions. Determining how DSBs are formed from such encounters and how these are repaired has been an important area of research for many years. Until recently, the prevailing hypothesis was that fork encounter of a SSB in the leading- strand template resulted in a single-ended DSB (seDSB) by “replication runoff”, whereas encounter of a lagging-strand template SSB could be successfully “skipped over” by the fork leaving behind a double-ended DSB (deDSB) in one of the replicated sister chromatids. However, recent work, using the Xenopus egg extract system to study replication fork-SSB collisions in-vitro, has shown that both leading and lagging template strand SSBs cause seDSBs. An important goal is to establish whether this new paradigm holds true in-vivo. Unlike deDSBs, seDSBs are thought to be repaired mainly by a highly mutagenic process termed break-induced replication (BIR). Our understanding of the molecular mechanism of BIR has come mainly from studies of the repair of non-S-phase associated DSBs in budding yeast. How well these reflect what happens during the repair of replication-associated DSBs, and whether BIR is well-conserved amongst eukaryotes, are largely unanswered questions.
Here, I establish for the first time the Flp-nick system in fission yeast. This experimental tool utilises a re-engineered version of the Flp recombinase to induce irreversible SSBs at Flp Recognition Target (FRT) sites inserted into the genome of interest. Following nick induction, the re-engineered Flp protein remains covalently attached to the 3’-end of the SSB via a phosphotyrosyl DNA linkage, essentially mimicking a trapped Topoisomerase 1. In my set-up, the FRT site is inserted at the ade6 locus on chromosome 3 of fission yeast, where DNA replication occurs almost always in the same direction. This allows us to designate whether the SSB is in the leading or lagging template strand based on the FRT site orientation. I use this system to study the recombination events generated following the encounter of the SSB in both leading and lagging template strands by the replication fork.
I show that a Flp-nick generated SSB does not affect viability in fission yeast. However, as expected, it does induce recombination in its immediate vicinity. It also induces template switching at a genetic reporter positioned “downstream” of the SSB, which is indicative of BIR. Importantly, both leading and lagging template strand SSBs induce template switching suggesting that both give rise to seDSBs upon fork encounter, which in turn lead to BIR. I confirm these findings using another system to induce site-specific SSBs, the M13 filamentous bacteriophage gpII protein. With both the Flp-nick system and gpII systems, I find that converging forks limit BIR-associated template switching. I confirm that template switching is dependent on Rad52 and requires Rad51, and I determine the importance of several candidate proteins on Flp- nick-induced recombination.

Keywords: break induced replication
flp-nick system
fission yeast
single stranded breaks in S-phase
Advisor: Whitby, M
Uphoff, S