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Title: Temporal control of translesion synthesis in vertebrates
Author: Edmunds, C. E.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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Translesion synthesis is a potentially mutagenic method of damage encountered by replication forks in the DNA template. The high-fidelity replicative polymerase can be replaced by a translesion polymerase which is better able to accommodate damaged or mismatched bases in its active site. Mutation occurs due to the non- or mis-instructional nature of the lesion and due to the naturally lower fidelity of these specialised polymerases. In the budding yeast Saccharomyces cerevisiae translesion synthesis is controlled by ubiquitination of the DNA sliding clamp PCNA, but in vertebrates the situation is more complex; PCNA ubiquitination is not epistatic to at least two translesion polymerases (Rev1 and DNA polymerase κ). Translesion synthesis can take place either at the replication fork, maintaining replication fork integrity, or replication can restart downstream of the lesion leaving a postreplicative gap that must be filled in later. I have examined these two scenarios separately, and found that they have different genetic requirements. By using stretched DNA fibres to monitor ongoing replication forks. I have shown that translesion synthesis at the replication fork requires Rev1 but not PCNA ubiquitination. This role of Rev1 in ‘on-the-fly’ translesion synthesis is mediated by its C-terminus, which contains the translesion polymerase interaction domain and the PCNA interaction domain. PCNA ubiquitination is, however, necessary for translesion synthesis at postreplicative gaps. Furthermore, two-hybrid analysis and in vitro pulldowns have shown that Rev1 can interact directly with PCNA without ubiquitination of PCNA, suggesting that it can act as an alternative adaptor between PCNA and the other translesion polymerases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available