Identification and functional characterisation of the POLD₃ subunit of DNA polymerase delta in Trypanosoma brucei

Abstract

Accurate and efficient genome replication is an essential process for all cellular life. In eukaryotes, three essential DNA polymerase complexes co-operate to replicate the nuclear genome: DNA polymerase α-primase (Pol α-primase), initiates DNA synthesis on both strands, by synthesising short RNA-DNA primers, from which DNA polymerase delta (Pol δ) and DNA polymerase epsilon (Pol ε) extend. Pol δ is the lagging strand polymerase, whilst both Pol δ and Pol ε have roles in leading strand replication. Pol δ is also the major polymerase for DNA synthesis during many DNA repair processes.

This study aimed to purify the previously uncharacterised Trypanosoma brucei Pol α-primase and Pol δ complexes, to determine their subunit composition, and identify potential druggable targets. T. brucei is the causative agent of potentially fatal African trypanosomiasis in humans and other mammals (sleeping sickness and Nagana respectively), for which current therapeutic options are limited by toxicity, complex administration and resistance.

Eukaryotic Pol δ is a heterotetramer in humans and fission yeast (PolD1- PolD4), but only a heterotrimer in budding yeast (PolD1-PolD3). In this study, the T. brucei Pol δ complex was purified as a heterotrimer comprised of POLD1, POLD2 and a previously unidentified POLD3 homologue. T. brucei POLD3 possesses two conserved C-terminal PCNA binding motifs and is capable of binding PCNA in vitro and in vivo. Unlike PolD3 from other eukaryotes, POLD3 does not interact with the repair polymerase Pol ζ.

PolD3 is essential for cell proliferation in fission yeast but not in budding yeast, although it is required for several DNA repair processes. RNAi knockdown of POLD3 in procyclic form T. brucei suggested that POLD3 is not an essential protein but is required for repair of phleomycin-induced DNA damage. Because DSB repair is key to VSG switching and immune evasion, POLD3 may be an attractive therapeutic target.

Date of Award	21 Jun 2017
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Stuart MacNeill (Supervisor) & Terry K Smith (Supervisor)