The role of the ATPase activities of the Rad51 and Rad54 proteins in the postsynaptic stage of homologous recombination

Abstract

Rad51 and Rad54, two DNA repair proteins which play central role in the homologous recombination (HR) pathway, both have DNA-dependent ATPase activities. Their respective ATPase activities have been shown previously to be critical for their in vivo function. On the other hand, exactly how their ATPase activities contribute to the in vivo function is still elusive. To understand the roles of their ATPase activities in HR, especially at the postsynaptic stage, Rad51 and Rad54 together with their ATPase-deficient mutant proteins, Rad51-K191R and Rad54-K341R respectively, were purified and characterized in vitro. Using nucleoprotein gel assays and a topological assay, we were able to show that the ATPase activities of both proteins are required for efficient dissociation of Rad51 protein fromdsDNA. Rad51 bound to dsDNA represents the product complex of DNA strand exchange. Additionally, salt-midpoint titration experiments showed that the Rad51-K191R mutant protein displayed DNA binding defects with both ssDNA and dsDNA, in addition to its strong defect in ATP-hydrolysis. Our results suggest that the ATPase activities of Rad51 and Rad54 both contribute to disassemble the Rad51-dsDNA product complex after DNA strand exchange, which may represent a critical function at the postsynaptic stage of HR pathway. This research is supported by NIH RO1 grant to W.-D. Heyer.