Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Protein-DNA Interactions

Background:

• RAD51 recombinase forms nucleoprotein filaments on single-stranded DNA (ssDNA) to mediate homologous recombination (HR) and replication fork restart.
• RAD51 filament activity is ATP-dependent, but non-productive ADP-bound or nucleotide-free states can impede DNA repair.
• The RAD51 paralog complex RFS-1/RIP-1 is involved in DNA repair pathways.

Purpose of the Study:

• To elucidate the mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 regulates RAD51 filament formation on ssDNA.
• To define how RFS-1/RIP-1 controls the nucleotide-bound state of RAD51 on ssDNA.
• To understand the role of RAD51 paralogs in ensuring the fidelity of HR by controlling RAD51 filament composition.

Main Methods:

• Biochemical assays to study RAD51 and RFS-1/RIP-1 interactions.
• Analysis of RAD51 filament formation and turnover on ssDNA in vitro.
• Characterization of nucleotide binding and hydrolysis states of RAD51.

Main Results:

• RAD51 paralogs (RFS-1/RIP-1) promote the dissociation of ADP-bound RAD51 from ssDNA.
• RFS-1/RIP-1 inhibits the binding of nucleotide-free RAD51 to ssDNA.
• In contrast, RAD51 paralogs stabilize productive ATP-bound RAD51 nucleoprotein filaments.

Conclusions:

• RAD51 paralogs possess 'nucleotide proofreading' activities that ensure the enrichment of active, ATP-bound RAD51 filaments on ssDNA.
• These activities are crucial for efficient homologous recombination (HR) and genome stability.
• RFS-1/RIP-1 acts as a critical regulator of RAD51 function by controlling filament nucleotide status.