Molecular dissection of PrimPol, a novel primase-polymerase involved in damage tolerance during DNA replication in eukaryotic cells

Keen, Benjamin A (2015) Molecular dissection of PrimPol, a novel primase-polymerase involved in damage tolerance during DNA replication in eukaryotic cells. Doctoral thesis (PhD), University of Sussex.

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Abstract

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.

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: 04 Jun 2015 14:42
Last Modified: 22 Jun 2017 09:55
URI: http://sro.sussex.ac.uk/id/eprint/54095

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