Genome duplication is an essential task our cells have to achieve prior to cell division, and requires a highly specialized replication machinery to ensure it is performed in an accurate and complete manner. DNA primase and polymerases are essential components of the replisome. Primases initiate DNA replication by synthesising short RNA primers that are then elongated by faithful and processive replicative DNA polymerases. However, both exogenous and endogenous agents can damage DNA and hinder progression of the replicative machinery. Translesion synthesis DNA polymerases assist in bypassing these DNA lesions in a process called DNA damage tolerance that enables chromosomal replication to proceed in in spite of damaged templates. This thesis details the characterisation of a novel eukaryotic DNA primase, coiled-coil domain containing protein (CCDC111), a member of the Archaeo Eukaryotic Primase (AEP) superfamily. Preliminary in vitro characterisation of CCDC111 demonstrated that the recombinant protein is capable of both DNA-dependant priming and polymerase activities, which is unprecedented for a eukaryotic polymerase, and it was therefore renamed Primase-polymerase (PrimPol). The aim of this thesis was to provide one of the first cellular characterisations of PrimPol by generating a knockout of the gene in avian DT40 cells and also depleting the protein in human cells using RNAi. In vivo evidence supports the involvement of this novel polymerase in replication fork progression following replicative stress, such as exposure to UV light, but also during unperturbed DNA replication. Work in this thesis also indicates a role for PrimPol in mitochondrial DNA maintenance. Together, the data presented here establish a role for PrimPol in DNA damage tolerance in avian and human cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577325 |
| Date | January 2013 |
| Creators | Bianchi, Julie |
| Publisher | University of Sussex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://sro.sussex.ac.uk/id/eprint/45544/ |
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