Comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for efficient pairing and strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. A comparison of the filament structures of both human and budding yeast Rad51 and Dmc1 proteins is presented here. Previous studies of the Dmc1 filament have described a structure distinct from that observed for other RecA-like protein filaments, including Rad51. The data presented here indicate that for all of the structural parameters examined, including persistence length, helical pitch, filament diameter, stoichiometry of DNA binding and helical handedness, Rad51 and Dmc1 filaments are nearly identical. In addition to the data indicating a high degree of structural conservation, the observation of calcium stimulation of budding yeast Rad51 recombinase activity demonstrates a higher degree of similarity between the in vitro activities of RecA-like recombinases than was previously known. As is the case for other recombinaes of this family, calcium stimulates yeast Rad51 recombinase activity by inhibiting ATP hydrolysis and increasing the average length of nucleoprotein filaments in the active conformation.; Stimulation of human Rad51 activity by the small molecule compound RS-1 is characterized here as well. Phillip Connell and Krishanthi Jayathilaka identified the compound RS-1 and described its ability to stimulate the DNA binding and strand assimilation activities of human Rad51 protein while not affecting the ATPase activity of the protein. This work further characterizes the effects of RS-1. The ultrastructural analysis of filaments formed in the presence of RS-1 demonstrate the ability of this compound to increase the average filament length and stabilize filaments in the active conformation. Although stabilization of the active conformation of Rad51 is usually associated with an inhibition of ATP hydrolysis, as with calcium stimulation, RS-1 can stabilize the presynaptic filament without affecting ATP hydrolysis.; The comparison of Dmc1 and Rad51 filament structures and the calcium experiments demonstrating similar in vitro activities, suggest that differences in the meiotic function of Rad51 and Dmc1 do not arise from intrinsic differences in filament structure or mechanism of activity, but rather as the result of unique regulation by accessory proteins during meiosis. Therefore, progress towards the purification of budding yeast Dmc1 and its interacting proteins from a normal meiotic context is presented here as well.; The in vivo significance of the structural and biochemical similarities of Dmc1 Rad51 and a possible mechanism for RS-1 stimulation of human Rad51 are discussed in detail.