Role of N-terminal domain of RAD54 in Branch Migration of Holliday Junctions

Abstract

Homologous recombination plays an important role in repairing the most harmful types of DNA damage, including DNA double-strand breaks and interstrand cross-links, in promoting faithful chromosome segregation during meiosis, and in telomere maintenance. RAD54 protein, a member of theSwi2/Snf2 family of ATP-dependent DNA translocases, is crucial to the homologous recombination pathway. The protein is conserved in all eukaryotes. RAD54 is a multifunctional protein that promotes branch migration of Holliday Junctions, chromatin remodeling, and stimulation of DNA strand exchange activity of RAD51. The structure of RAD54 lacking the N-terminal domain has been solved. The central domain of RAD54 has DNA dependent ATPase motifs that are well conserved in all members of helicase Superfamily 1 and 2; however, the N-terminal domain is unique for RAD54 orthologues and is not well understood. Here, using biochemical approaches and site-directed mutagenesis, we characterized the role of the RAD54 N-terminal domain in branch migration of Holliday junctions. We found that by removing N-terminal domain residues we were able to uncouple ATPase and branch migration activities of RAD54. Also, we found that a recently identified DNA binding site in the N-terminal domain of RAD54 has a preference for branched DNA substrates similar to that of the full length RAD54. Disruption of DNA binding of the N-terminal domain inhibited the branch migration activity of RAD54 without affecting its ATPase activity. Our results demonstrate for the first time that the DNA binding by the N-terminal domain is necessary for the branch migration activity of RAD54. This requirement of second DNA binding domain for branch migration may be conserved among branch migration proteins. We also identified specific small-molecule inhibitors for branch migration activity of RAD54. Interestingly, the initial analysis showed that two smallmolecule compounds, C-A23, and C-G01 had a stronger inhibition on branch migration without significantly affecting its ATPase activity. This suggests that they may inhibit branch migration by interacting with the N-terminal domain of RAD54. However, further work needs to done to determine their exact mechanism of action and the function of branch migration in cells using these inhibitors.%%%%Ph.D., Biochemistry – Drexel University, 2017