The Shu complex is an ATPase that regulates Rad51 filaments during homologous recombination in the DNA damage response.

Rad51 filaments are Rad51-coated single-stranded DNA and essential in homologous recombination (HR). The yeast Shu complex (Shu) is a conserved regulator of homologous recombination, working through its modulation on Rad51 filaments to direct HR-associated DNA damage response. However, the biochemical properties of Shu remain unclear, which hinders molecular insights into Shu's 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 fork-shaped DNA with 20nt ssDNA components. 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 and alters the properties of ssDNA and the filaments with a 5'-3' polarity. The alterations depend on the ATP hydrolysis of Shu, suggesting that the ATPase activity of Shu is important in regulating its functions. 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.