ABSTRACT
The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.
Subject(s)
BRCA2 Protein/genetics , DNA Replication , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , RNA-Binding Proteins/genetics , Rad51 Recombinase/genetics , Adenosine Triphosphate/metabolism , BRCA2 Protein/metabolism , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Hydrolysis , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , RNA-Binding Proteins/metabolism , Rad51 Recombinase/metabolism , Signal Transduction , Single Molecule Imaging , Red Fluorescent ProteinABSTRACT
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.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , RNA Editing , Rad51 Recombinase , Humans , Adenosine Triphosphate/metabolism , DNA Replication/genetics , DNA, Single-Stranded , Gene Editing , Genomic Instability/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolismABSTRACT
Genomic integrity depends on the RecA/RAD51 protein family. Discovered over five decades ago with the founder bacterial RecA protein, eukaryotic RAD51 is an ATP-dependent DNA strand transferase implicated in DNA double-strand break and single-strand gap repair, and in dealing with stressed DNA replication forks. RAD51 assembles as a nucleoprotein filament around single-stranded DNA to promote homology recognition in a duplex DNA and subsequent strand exchange. While the intrinsic dynamics of the RAD51 nucleoprotein filament has been extensively studied, a plethora of accessory factors control its dynamics. Understanding how modulators control filament dynamics is at the heart of current research efforts. Here, we describe recent advances in RAD51 control mechanisms obtained specifically using fluorescence-based single-molecule techniques.
Subject(s)
Nucleoproteins , Rad51 Recombinase , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , DNA, Single-Stranded/genetics , Nucleoproteins/genetics , Nucleoproteins/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolismABSTRACT
Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.