Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Damasceno, J. D., Reis-Cunha, J., Crouch, K. , Beraldi, D., Lapsley, C., Tosi, L. R.O., Bartholomeu, D. and McCulloch, R. (2020) Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication. PLoS Genetics, 16(7), e1008828. (doi: 10.1371/journal.pgen.1008828) (PMID:32609721) (PMCID:PMC7360064)

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Abstract

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Crouch, Dr Kathryn and Beraldi, Dr Dario and Damasceno, Dr Jeziel and Lapsley, Mr Craig and McCulloch, Professor Richard
Creator Roles:
Damasceno, J. D.Conceptualization, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing
Crouch, K.Data curation, Methodology, Resources, Software, Visualization, Writing – review and editing
Beraldi, D.Formal analysis, Methodology, Software, Visualization, Writing – review and editing
Lapsley, C.Formal analysis, Investigation, Validation, Writing – review and editing
McCulloch, R.Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing
Authors: Damasceno, J. D., Reis-Cunha, J., Crouch, K., Beraldi, D., Lapsley, C., Tosi, L. R.O., Bartholomeu, D., and McCulloch, R.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Journal Name:PLoS Genetics
Publisher:Public Library of Science
ISSN:1553-7390
ISSN (Online):1553-7404
Copyright Holders:Copyright © 2020 Damasceno et al.
First Published:First published in PLoS Genetics 16(7): e1008828
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
173173How do common and diverged features of the replicative stress response shape the biology of TriTrypRichard McCullochBiotechnology and Biological Sciences Research Council (BBSRC)BB/N016165/1III - Parasitology
301963Does genome replication in the protozoan parasite Leishmania rely on origin-indepenent initiation?Richard McCullochBiotechnology and Biological Sciences Research Council (BBSRC)BB/R017166/1III - Parasitology
170547The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOTR)104111/Z/14/ZRIII - Parasitology