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Title: Investigating the molecular mechanism of replication restart in fission yeast
Author: Nguyen, Michael Ong
ISNI:       0000 0004 5354 5872
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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Successful replication of the genome during each cell cycle requires that every replication fork merge with its opposing fork. However, lesions in the template DNA or protein-DNA barriers often impede replication forks and threaten the timely completion of genome duplication. If a fork encounters a replication fork barrier (RFB), it can be subject to a variety of fates. In some cases the replisome is maintained in a manner such that it can resume DNA synthesis when the barrier is removed. Alternatively the stalled fork is simply held in a competent state to merge with the opposing fork when it arrives. However, fork stalling can also precipitate dissociation of the replisome (fork collapse) or even fork breakage. If this happens the recombination machinery can intervene to restore DNA integrity and restart replication, albeit with a risk of causing deleterious genetic change if ectopic homologous sequences are recombined. I have exploited a site-specific RFB in fission yeast termed RTS1 to investigate the consequences of perturbing a single replication fork. RTS1 is a polar RFB (i.e. it blocks fork progression in a unidirectional fashion), enabling replication to be completed by the opposing fork. Despite this, fork blockage at RTS1 triggers a strong recombinational response that is able to restart DNA synthesis, which at least initially is highly error prone. Here, I present my work in establishing a live cell imaging approach to visualizing the recombinational response at the RTS1 RFB, demonstrating that the majority of cells initiate recombination-dependent replication (RDR). RDR begins within a few minutes of fork blockage and is only curtailed by the arrival of the opposing fork. It depends on the Rad52 protein, which remains associated with the restarted fork and whose presence correlates with its infidelity. I also illustrate the significance of various genetic factors, including Rad51, the Rad51 mediators, Fml1 helicase, Rad54 translocase, Pfh1 sweepase, and Cds1 checkpoint kinase, in modulating Rad52 localization and block-induced recombination at the RTS1 RFB.
Supervisor: Whitby, Matthew Conway Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; DNA replication ; homologous recombination ; replication fork barrier