The trimeric Shu complex in C. elegans is an ATPase that remodels RAD51 filaments in the homologous recombination-associated DNA damage response

Homologous recombination (HR) is critical to error-free lesion bypass in the DNA damage response. This error-free lesion tolerance pathway is initiated by the RAD51 recombinase, which forms nucleoprotein filaments on single-stranded DNA (ssDNA) to facilitate template-directed repair using homologous sister chromatids. RAD51 filaments are tightly regulated by RAD51 mediator proteins. Among these, the Shu complexes facilitate HR-directed DNA damage tolerance and are evolutionarily conserved from yeast to humans. The Caenorhabditis elegans Shu complex is a heterotrimer consisting of three protein subunits: RFS1, RIP1, and SWS1. However, the biochemical properties of this trimeric complex remain unclear. Here, we report the biochemical characterization of the C . elegans Shu complex and its interactions with DNA, ATP, and Rad51 filaments. We first revealed that the Shu trimer preferentially binds DNA with an exposed 5′ end, particularly favoring a fork-shaped double-stranded DNA (dsDNA). Then, we found that the trimer binds to ATP and possesses DNA-dependent ATPase activity. Through site-specific mutagenesis, we identified the catalytic residues in the RFS1 domain and validated the ATPase activity. Using fluorescence-based assays, we further demonstrated that the Shu trimer remodels RAD51 filaments in an ATP-hydrolysis-dependent manner and stabilizes the filaments in an ATP-binding-dependent manner. These findings provide key mechanistic insights into how the C . elegans Shu complex regulates RAD51 filaments, priming them for downstream HR-mediated DNA repair processes.