Timely completion of DNA replication in each cell cycle is crucial for maintaining genomic integrity. This is often challenged by the presence of various replication fork barriers (RFBs). On collision with a RFB, the fate of the replication fork remains uncertain. In some cases, the integrity of the fork is maintained until the barrier is removed or the fork is rescued by merging with the incoming fork. However, fork stalling can cause dissociation of all of the associated replication proteins (fork collapse). If this occurs, the cell's recombination machinery can intervene to help restart replication in a process called recombination-dependent replication (RDR). Programmed protein-DNA barriers like the Replication Terminator Sequence-1 (RTS1) have been used to demonstrate that replication fork blockage can induce recombination. However, it remains unclear how efficiently this recombination gives rise to replication restart and whether the restarted replication fork exhibits the same fidelity as an origin-derived fork. It is also unknown whether accidental replication barriers induce recombination in the same manner as programmed barriers. In this study, I introduce recombination reporters at various sites downstream of RTS1 to obtain information on both the fidelity and efficiency of replication restart. I find that unlike break induced replication (BIR), the restarted fork gives rise to hyper-recombination at least 75 kb downstream of the barrier. Surprisingly, fork convergence, rather than inducing recombination, acts to prevent or curtail genetic instability associated with RDR. I also investigate a number of genetic factors that have a role in either preventing or promoting genome instability associated with the progression of the restarted fork. To compare RTS1 with an accidental protein-DNA barrier, a novel site-specific barrier system (called MarBl) was established based on the human mariner transposase, Hsmar1, binding to its transposon end. Replication fork blockage at MarBl strongly induces recombination, more so than at RTS1. This appears to be a general feature of accidental barriers as introduction of the E. coli TusB-TerB site-specific barrier in S. pombe gives rise to a similar effect. Here, I compare and contrast accidental barriers with programmed barriers. I observe that there is very little replication restart, if any, at MarBl measured by direct repeat recombination downstream. This points to the fact that accidental barriers do not trigger fork collapse in the same way as programmed RFBs and that the increased recombination that they cause may be a consequence of the inability of replication forks to terminate correctly, owing to the bi-directional nature of the barrier. Several genetic factors are assessed for their impact on MarBl-induced recombination, which further highlights both similarities and differences with RTS1-induced recombination.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:730176 |
| Date | January 2016 |
| Creators | Jalan, Manisha |
| Contributors | Whitby, Matthew |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:aed1673a-f967-41a5-9643-2e432052e174 |
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