Genome instability induced by structured DNA and replication fork restart

Schalbetter, Stephanie (2012) Genome instability induced by structured DNA and replication fork restart. Doctoral thesis (DPhil), University of Sussex.

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DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if replication resumes or restarts in these circumstances in order to complete genome replication is not well understood and the fidelity of replication in response to such obstacles remains unclear. I have developed TR assays to assess replication errors in the context of replication fork restart and secondary structures. The results suggest that structured DNA (G4) can cause instability of TRs in the context of normal replication and that restarted replication can be intrinsically error-prone. Surprisingly, the mutagenic effect of G4-DNA on TR stability was not elevated in the context of replication fork restart. Therefore, deletions of TRs containing G4-DNA are not more susceptible to the compromised fidelity of a restarted replication fork.

Structures such as stalled replication forks can induce checkpoint responses to maintain genome stability. The stabilisation of replication forks is central in the response to replication stress. These protective mechanisms include the regulation of enzymatic activities. Mus81-Eme1 is a structurespecific endonuclease which is regulated by the DNA replication checkpoint, but has also been shown to be required for replication fork restart in certain circumstances. In collaboration with Professor Neil McDonald I analysed a novel domain identified in Mus81-Eme1. Mutagenesis of key residues deduced from the protein structure and comparison of their genetic analysis to known phenotypes of Mus81-Eme1 suggests distinct requirements for this domain.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics
Depositing User: Library Cataloguing
Date Deposited: 18 May 2012 08:42
Last Modified: 25 Aug 2015 14:01

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