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Title: Molecular dissection of PrimPol, a novel primase-polymerase involved in damage tolerance during DNA replication in eukaryotic cells
Author: Keen, Benjamin A.
ISNI:       0000 0004 5347 914X
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 2015
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PrimPol is a recently identified member of the archaeo-eukaryotic primase (AEP) family of proteins. It possesses both primase and polymerase activities and is involved in the replication of both nuclear and mitochondrial DNA. PrimPol is predicted to possess an AEP polymerase and a UL52-like zinc finger domain. This thesis establishes the roles of these domains in the context of PrimPol's catalytic activities. Although apparently dispensable for polymerase activity, the zinc finger is essential for maintaining primase activity and also appears to play an important role in regulating the processivity and fidelity of PrimPol's extension activities. A recently study identified a PrimPol mutation (Y89D) that is potentially associated with the development of high myopia in humans. Here, the biochemical defects associated with this mutant are analysed and described. This protein variant has a significant reduction in polymerase activity. Mutational analysis suggests that the hydrophobic ring of tyrosine is important for retaining wildtype DNA extension activity. Biophysical analysis of the secondary structure and stability of this PrimPol variant suggests that this PrimPol variant has reduced α-helical content and is less stable than the wild-type protein. Finally, the interaction of PrimPol with single-stranded DNA binding protein replication protein A (RPA) is investigated. Previous studies have identified an interaction of PrimPol with RPA. Here, it is demonstrated that PrimPol has two separate RPA interaction motifs and a crystal structure is presented of one such motif in PrimPol bound to RPA that reveals the molecular basis for this interaction. Together, these studies provide molecular insights into the catalytic mechanism of PrimPol as well as some of the key intramolecular and intermolecular mechanisms of that regulate the activities of PrimPol.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD0415 Biochemistry ; QH0426 Genetics