Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

• MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA, Single-Stranded * metabolism   genetics   MeSH
• Humans MeSH
• Multiprotein Complexes MeSH
• Mutation MeSH
• Rad51 Recombinase * metabolism   genetics   MeSH
• DNA Replication * genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• DNA, Single-Stranded * MeSH
• Multiprotein Complexes MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase * MeSH

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

• Keywords
• Genetics, Molecular biology, Molecular interaction, Properties of biomolecules,
• Publication type
• Journal Article MeSH

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

• MeSH
• Adenosine Diphosphate pharmacology   MeSH
• Adenosine Triphosphate pharmacology   MeSH
• Caenorhabditis elegans metabolism   MeSH
• Species Specificity MeSH
• Fluorescence MeSH
• Interferometry MeSH
• DNA, Single-Stranded metabolism   MeSH
• Nucleotides metabolism   MeSH
• Caenorhabditis elegans Proteins metabolism   MeSH
• Rad51 Recombinase chemistry   metabolism   MeSH
• Sequence Homology, Amino Acid * MeSH
• Protein Stability drug effects   MeSH
• Protein Binding drug effects   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Diphosphate MeSH
• Adenosine Triphosphate MeSH
• DNA, Single-Stranded MeSH
• Nucleotides MeSH
• Caenorhabditis elegans Proteins MeSH
• Rad51 Recombinase MeSH