CRISPR-dependent Base Editing Screens Identify Separation of Function Mutants of RADX with Altered RAD51 Regulatory Activity.
DNA damage
DNA replication
Genome stability
Replication stress
single-stranded DNA binding protein
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
01 10 2023
01 10 2023
Historique:
received:
19
06
2023
revised:
01
08
2023
accepted:
04
08
2023
pmc-release:
01
10
2024
medline:
15
9
2023
pubmed:
13
8
2023
entrez:
12
8
2023
Statut:
ppublish
Résumé
RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability. Here, we utilize a CRISPR-dependent base editing screen to tile mutations across RADX to delineate motifs required for RADX function. We identified separation of function mutants of RADX that bind DNA and RAD51 but have a reduced ability to stimulate its ATP hydrolysis activity. Cells expressing these RADX mutants accumulate RAD51 on chromatin, exhibit replication defects, have reduced growth, accumulate DNA damage, and are hypersensitive to DNA damage and replication stress. These results indicate that RADX must promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.
Identifiants
pubmed: 37572935
pii: S0022-2836(23)00346-7
doi: 10.1016/j.jmb.2023.168236
pmc: PMC10530557
mid: NIHMS1924164
pii:
doi:
Substances chimiques
Adenosine Triphosphate
8L70Q75FXE
DNA, Single-Stranded
0
RAD51 protein, human
EC 2.7.7.-
Rad51 Recombinase
EC 2.7.7.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
168236Subventions
Organisme : NCI NIH HHS
ID : P01 CA092584
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM116616
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM118089
Pays : United States
Informations de copyright
Copyright © 2023 Elsevier Ltd. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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