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Title: Investigating the molecular mechanisms of recombination-dependent replication in fission yeast
Author: Ishnazarova, Nadiya
ISNI:       0000 0004 7653 5757
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Perturbation of DNA replication is a major cause of cancer and other pathological states causing loss of genome integrity and catastrophic chromosomal rearrangements. To guard against such disaster, mechanisms exist to process and stabilize replication forks (RFs) blocked at barriers, restart collapsed forks and ensure faithful fork merging and termination. Despite recent advances, our understanding of the molecular mechanisms that drive recombination-dependent replication at the replication fork barriers (RFBs) is far from comprehensive, and in particular we know very little about mechanisms used to govern its accuracy. To characterize factors that are needed for the successful processing and restart of blocked RFs at the site-specific RFB Replication Termination Sequence RTS1 in Schizosaccharomyces pombe, I focused on studying a putative partner protein of the Fml1 DNA helicase called Dbl2; its orthologs can be found from yeast to human. I used a combination of yeast genetics, fluorescent microscopy and live cell imaging to investigate the role of Dbl2. Data from epistasis analysis, with key components of the homologous recombination machinery, highlights differences with a fml1∆ mutant and suggest that Dbl2 is involved in destabilizing the Rad51-nucleoprotein filament in conjunction with the Fbh1 DNA helicase. However, this genetic analysis also revealed that Dbl2 has functions that are independent of Fbh1 in the regulation of Rad51-independent Rad52-dependent recombination, spontaneous and induced at RTS1. This may include a role in and/or regulating other helicases such as Fml1 and Pfh1. Structure-function studies using a series of truncation and internal deletion mutants of Dbl2 demonstrate that the cooperative action of all regions is required for Dbl2 function to limit recombination induced at RF blocked at RTS1, to counteract Rad51 activity in the presence of Rad55 and for its nuclear localization to the multiple sites across the genome. However, only its middle region is important to confer resistance to genotoxins and genetic interaction with most Rad51 mediator proteins. In addition, I have developed expression and purification strategies for full-length Dbl2 and its functional domain and conducted preliminary experiments for the characterization of the conserved N-terminal domain DUF2439 structure.
Supervisor: Whitby, Matthew Sponsor: OCRC ; Cancer Research UK ; Clarendon Fund ; Somerville College Graduate Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available