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CRISPR-dependent base editing screens identify separation of function mutants of RADX with altered RAD51 regulatory activity
Open accessPreprint — published as 10.1016/j.jmb.2023.168236 · Paper page
Abstract
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 bind RAD51 and promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.
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Metadata
- Authors
- Adolph, M. B., Garje, A. S., Balakrishnan, S., Morati, F., Modesti, M., Chazin, W. J., & Cortez, D.
- Affiliations
- Vanderbilt University (US)
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Cortez, David
- ORCID
- Venue
- Journal of Molecular Biology
- Publisher
- openRxiv
- Published
- 2023-06-19
- Volume
- 435
- Issue
- 19
- Pages
- 168236-168236
- ISSN
- 0022-2836 (ISSN-L)
- PMC
- PMC10530557
- OpenAlex
- W4385738001
- Cited by
- 4
- References
- 2
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 2
- FWCI
- 0.36
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Molecular Biology (acceptedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- HAL (submittedVersion)
- Funders
- Ligue Contre le Cancer
- National Institutes of Health
- National Cancer Institute (P01CA092584)
- National Institute of General Medical Sciences (R01GM116616, R35GM118089)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- RADx, RAD51, single-stranded DNA binding protein, separation-of-function mutants, homologous recombination, replication fork protection, replication stress, DNA damage sensitivity, genome stability, DNA-binding motifs
- MeSH terms
- Gene Editing, Adenosine Triphosphate, DNA Replication, DNA, Single-Stranded, Humans, RNA Editing, Genomic Instability, Rad51 Recombinase, Clustered Regularly Interspaced Short Palindromic Repeats
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