The Role of Helicases and Helicase-like Proteins in Homologous Recombination

Homologous recombination is important for repair of the most harmful types of DNA damage including DNA double-strand breaks, interstrand cross-links, and for chromosome segregation in meiosis. When a double-strand break occurs, the DNA ends are resected to create single-stranded overhangs that allow RAD51 recombinase to form a nucleofilament that searches for homologous DNA to initiate repair. Once the sequence is found, RAD51 exchanges DNA between the homologous pairs forming a Holliday junction at the point of exchange. The translocation of the Holliday junction by branch migration and resolution results in completion of repair. Several helicases are involved in homologous recombination, but their functions are not well understood. This study focuses on FANCJ helicase and RAD54 helicase-like protein that are highly conserved in eukaryotes. Mutations in either protein can lead to disease and cancer. FANCJ is implicated in interstrand cross-link repair but its specific function is unknown. Here, we characterized FANCJ's ability to clear RAD51 from both single-stranded and double-stranded DNA which can potentially have a regulatory function in homologous recombination. For the first time we identified a FANCJ mutation that disrupts the coupling of ATPase activity with its ability to translocate on DNA and disrupt RAD51-single-stranded DNA complexes. We then investigated the role of the RAD54 ATPase activity in stimulation of RAD51 strand exchange. We identified a small molecule streptonigrin that specifically binds RAD54 and inhibits its ATPase. Surprisingly, we found that RAD54's ability to stimulate RAD51 DNA strand exchange was not significantly affected by streptonigrin indicating a minor role of RAD54 ATPase in this process. In contrast, as expected streptonigrin strongly inhibited RAD54 branch migration activity. Our results demonstrate for the first time that RAD54 ATPase activity plays a different role in stimulation of DNA strand exchange than in branch migration of Holliday junctions. Furthermore, isolating discrete functions through chemical inhibition as well as the basic characterization of homologous proteins are critically important for understanding their innate functions.