Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.
Journal
PLoS genetics
ISSN: 1553-7404
Titre abrégé: PLoS Genet
Pays: United States
ID NLM: 101239074
Informations de publication
Date de publication:
07 2020
07 2020
Historique:
received:
15
11
2019
accepted:
05
05
2020
revised:
14
07
2020
pubmed:
2
7
2020
medline:
9
9
2020
entrez:
2
7
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 32609721
doi: 10.1371/journal.pgen.1008828
pii: PGENETICS-D-19-01903
pmc: PMC7360064
doi:
Substances chimiques
Rad51 Recombinase
EC 2.7.7.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1008828Subventions
Organisme : Medical Research Council
ID : MR/S019472/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : G0401553
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R017166/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/N016165/1
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 104111
Pays : United Kingdom
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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