Searching for regulators of replicative helicase assembly in African trypanosomes

Abstract

Appropriate timing of DNA replication is fundamental for maintaining the stability of the genome. One of the key steps in regulating this timing in eukaryotes is the assembly of the replicative helicase, a multi-protein complex conserved throughout eukaryotes. This assembly is mediated by loading factors that act as intermediates between the cell cycle regulatory network and the replicative helicase. These loading factors have diverged in eukaryote evolution, a feature that could prove invaluable in the development of drugs against the human and animal infectious parasite Trypanosoma brucei.

Here, proximity labelling was employed in an effort to identify the loading factors of the replicative helicase in T. brucei, without success. However, this work explores the limitations of different proximity labelling approaches in detail. For one, the peroxidase- based (APEX2) proximity labelling cannot be used in T. brucei due to an endogenous factor catalysing the equivalent labelling reaction. Also, the first-generation promiscuous biotin ligases (BirA* and BioID2) are not sufficiently active to identify low abundance or temporal protein-protein interactions.

Yeast Dpb11 and human TopBP1 are replicative helicase loading factors that possess multiple BRCA1 C-terminal (BRCT) domains. This work explored the cellular and molecular function of two previously uncharacterised multi-BRCT domain proteins of T. brucei – KMBP1 and KMBP2. Since KMBP1 is not an essential protein, based on the viability of complete knock-outs of the gene, it is unlikely to be a functional homologue of Dpb11/TopBP1. Even though the N-terminal pair of KMBP1 BRCT domains is structurally similar to equivalent domains of Dpb11/TopBP1 as determined by X-ray crystallography. Nonetheless, we characterise KMBP1 as having a key role in DNA repair and present the first ever structure of a kinetoplastid BRCT domain. While we were unable to establish a function for KMBP2, evidence suggests that it is an essential protein.

Date of Award	29 Jul 2020
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Stuart MacNeill (Supervisor) & Terry K Smith (Supervisor)