Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca2+ and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. Delta G(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased Delta G(diss). Adding only ATP and Ca2+ to the buffer disintegrated the self-associated rods into rings and short helices of further increased Delta G(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K-d similar to 2 x 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the Delta G(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role.