Towards a structural understanding of DNA polymerase δ using the thermophilic fungus Chaetomium thermophilum as a model

Abstract

Eukaryotic DNA polymerase δ plays a critical role in DNA replication and repair. At the replication fork, Pol δ carries out the bulk of lagging strand DNA synthesis and plays transient but crucial roles in leading strand DNA replication as well. Human Pol δ consists of a catalytic subunit (PolD1) and three accessory subunits (PolD2, PolD3 and PolD4). The regulated degradation of PolD4 in response to DNA damage reflects the important role of this protein, however, its precise function remains to be fully understood. Structural studies have located the PolD4 protein between PolD1 and PolD2, leading to the suggestion that PolD4 degradation is likely to bring about a subtle change in the three-dimensional architecture of Pol δ to modulate the enzyme’s activity.
This work used PolD4 proteins from fission yeast Schizosaccharomyces pombe and thermophilic fungus Chaetomium thermophilum (Ct) as models to explore the function and structure of PolD4. Overexpression of S. pombe PolD4 could supress mutations in genes encoding PolD1, PolD2 and PolD3. We investigated how the conserved residues at the C-terminal domain of PolD4 contribute to this function. We identified a PCNA interacting protein (PIP) motif in the N-terminal region of CtPolD4 and solved the crystal structure of CtPolD4 PIP peptide bound to CtPCNA. This structure reveals a non-canonical mode of binding that contrasts with the recently characterised human PolD4 (p12) PIP-PCNA interaction.
As part of a wider study dissecting the structure and function of Pol δ, we developed protocols for expression and purification of CtPol δ and obtained its cryo-EM structure to near 4 Å. Our studies may provide insights on the role of PolD4 in contribution to Pol δ function, regulation of DNA replication and maintenance of genome stability.

Date of Award	15 Jun 2022
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Stuart MacNeill (Supervisor)