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.

Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state

“…Statistical analysis of RAD51 WT incubated with ATP showed an average filament length of 71 nm ( Supplementary Figure S1B ). This corresponds to the theoretical length of a 150-nt ssDNA extended by a factor of ∼1.5 compared to the length of B-form DNA ( 19 ) and is in agreement with previously published data for RAD51 filaments ( 23 ). Accordingly, the peak maximum filament length observed for RAD51 WT in the presence of AMP-PNP as well as for the KR and KA mutants lies between ∼39 and 44 nm ( Supplementary Figure S1B ), further supporting compressed ‘closed’ properties of these filaments.…”

New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability

“…The RAD51 protein presents different oligomeric forms in buffer solution [35,36]. Structure resolutions of RAD51 in different organisms show that it is frequently organized in a ring with 6 to 8 monomers [37,38], and even in short filaments depending on the buffer composition [39]. The equilibrium between these different forms is also dependent on the protein concentration [35] and the presence of other molecules like peptides, salt or co-factors.…”

“…Nucleotide cofactors are also important elements to consider as they modulate the equilibrium and configuration of RAD51 oligomers. Schay et al [39] demonstrate the role of ATP in the specific conformation of RAD51. By electron microscopy, they show that the binding of ATP modifies the protein-protein interfaces and leads mainly to oligomeric structures in the form of rings or short helices.…”

“…The ring structure was predominant when the salt concentration was increased. All our experiments were carried out in saturating concentrations of ATP (2 mM) in order to avoid the formation of disordered filaments as described by Shay et al [39]. Moreover, the biologically active oligomer form requires ATP [41].…”

Chromosome behavior and the molecular basis of meiosis

“…另外, 一些参与DSB修复的DNA损伤应答(DNA damage response, DDR)蛋白, 如BRCA1(breast cancer type 1 susceptibility protein), MDC1(mediator of DNA damage checkpoint protein 1)和53BP1(transformation related protein 53 binding protein 1) [51] 等也被发现在体细胞RAD51 的募集过程中有重要作用. 最近, Schay等人 [52] 用物 理化学的方法证明, 人类RAD51必须与ATP结合后才 能被募集到单链DNA上. 此外, 有报道称, 在体细胞 中RAD51的募集受其磷酸化的影响 [53] , 但其实验结…”