title: Without Binding ATP, Human Rad51 Does Not Form Helical Filaments on ssDNA.
abstract: 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 Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(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) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(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.
authors.affiliation: Department of Biophysics and Radiation Biology, Semmelweis University , Tűzoltó utca 37-47, Budapest H-1094, Hungary.
authors.forename: Gusztáv
authors.initials: G
authors.lastname: Schay
authors.affiliation: Department of Biophysics and Radiation Biology, Semmelweis University , Tűzoltó utca 37-47, Budapest H-1094, Hungary.
authors.forename: Bálint
authors.initials: B
authors.lastname: Borka
authors.affiliation: MTA-ELTE NAP B Neuroimmunology Research Group, Department of Biochemistry, Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest H-1117, Hungary.
authors.forename: Linda
authors.initials: L
authors.lastname: Kernya
authors.affiliation: MTA-ELTE NAP B Neuroimmunology Research Group, Department of Biochemistry, Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest H-1117, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Bulyáki
authors.affiliation: MTA-ELTE NAP B Neuroimmunology Research Group, Department of Biochemistry, Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest H-1117, Hungary.
authors.forename: József
authors.initials: J
authors.lastname: Kardos
authors.affiliation: Department of Biophysics and Radiation Biology, Semmelweis University , Tűzoltó utca 37-47, Budapest H-1094, Hungary.
authors.forename: Melinda
authors.initials: M
authors.lastname: Fekete
authors.affiliation: Department of Biophysics and Radiation Biology, Semmelweis University , Tűzoltó utca 37-47, Budapest H-1094, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Fidy
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase