Shu complex is an ATPase that regulates Rad51 filaments in homologous recombination-directed DNA damage response.

Rad51 filaments are Rad51-coated single-stranded DNA and essential intermediates in homologous recombination (HR) and the HR-associated DNA damage response. The yeast Shu complex (Shu) is a conserved regulator of HR, working through its modulation of Rad51 filaments. However, the biochemical properties of Shu remain unclear, which hinders molecular insight into Shus role in HR and the DNA damage response. In this work, we biochemically characterized Shu and analyzed its molecular actions on single-stranded DNA and Rad51 filaments. First, we revealed that Shu preferentially binds DNA with ssDNA components and ssDNA/double-stranded DNA junctions. Then, we identified and validated, through site-specific mutagenesis, that Shu is an ATPase and hydrolyzes ATP in a DNA-dependent manner. Furthermore, we showed that Shu interacts with ssDNA and Rad51 filaments at the 5 end preferentially, altering the conformations of ssDNA and the filaments. The alterations depend on the ATP hydrolysis of Shu, suggesting that the ATPase activity of Shu is important in regulating its functions in HR. The preference of Shu for acting on the 5 end of Rad51 filaments aligns with the observation that Shu promotes lesion bypass at the lagging strand of a replication fork. Our work on Shu, a prototype modulator of Rad51 filaments in eukaryotes, provides a general molecular mechanism for Rad51-mediated error-free DNA lesion bypass.