Real-time measurements of the nucleation, growth and dissociation of single Rad51–DNA nucleoprotein filaments

Abstract

Human Rad51 (hRad51), the protein central to DNA pairing and strand exchange during homologous recombination, polymerizes on DNA to form nucleoprotein filaments. By making use of magnetic tweezers to manipulate individual DNA molecules, we measured the nucleation and growth of hRad51 nucleoprotein filaments, and their subsequent disassembly in real time. The dependence of the initial polymerization rate upon the concentration of hRad51 suggests that the rate-limiting step is the formation of a nucleus involving 5.5 ± 1.5 hRad51 monomers, corresponding to one helical turn of the hRad51 nucleoprotein filament. Polymerization is highly cooperative (i.e. a nucleation-limited reaction) at low concentrations and less cooperative (a growth-limited reaction) at high concentrations of the protein. We show that the observed preference of hRad51 to form nucleoprotein filaments on double-stranded DNA rather than on single-stranded DNA is due to the fact that it depolymerizes much faster from ssDNA than from dsDNA: indeed, hRad51 polymerizes faster on ssDNA than on dsDNA. Hydrolysis of ATP by hRad51 does not correlate with its dissociation from dsDNA. This suggests that hRad51 does not depolymerize rapidly from dsDNA after strand exchange but stays bound to the heteroduplex, highlighting the importance of partner proteins to facilitate hRad51 depolymerization from dsDNA.

Direct observation of twisting steps during Rad51 polymerization on DNA

“…We observed that the polymerization of hRad51 on dsDNA requires a minimal concentration of approximately 100 nM to initiate. This is consistent with previous results independently obtained both with conventional MT (20) and in single-molecule videomicroscopy (21) confirming the existence of a threshold concentration for hRad51 polymerization initiation. The threshold in ref.…”

“…This contrasts with our previous report (20) in which the magnetic bead was blocked in torsion by the MT and the DNA molecule untwisted manually. We attribute this behavior to the viscous drag of the bead, which opposes the formation of the nucleoprotein filament and becomes the limiting factor at high hRad51 concentrations.…”

“…We previously observed (20) that nonhydrolyzable ATP analogs considerably slowed down the polymerization in conventional MT, suggesting that the association energy is weaker with these cofactors, or the association energy barrier higher. It is thus possible that polymerization of hRad51 in the presence of a nonhydrolyzable ATP analog is too small to overcome the torque induced by the residual horizontal magnetic field of the FRMT [Warning: the rotation seems faster at 10 nM, but averaging polymerization speed on such a short period is meaningless owing to the strong polymerization speed fluctuations apparent in A (see text).]…”

“…Reported values range from 3 to 7 bp per protein for RecA (5,(7)(8)(9)) and 2 to 3 and 4 to 5 bp per protein for Rad51 (4,(10)(11)(12). The kinetics of RecA and Rad51 polymerization were recently the subject of several singlemolecule studies (13)(14)(15)(16)(17)(18)(19)(20)(21). There is consensus about a nucleation and growth mechanism for both RecA (14)(15)(16)22) and Rad51 (19)(20)(21), and about a highly cooperative nucleation step requiring the simultaneous binding of several proteins.…”

“…The kinetics of RecA and Rad51 polymerization were recently the subject of several singlemolecule studies (13)(14)(15)(16)(17)(18)(19)(20)(21). There is consensus about a nucleation and growth mechanism for both RecA (14)(15)(16)22) and Rad51 (19)(20)(21), and about a highly cooperative nucleation step requiring the simultaneous binding of several proteins. The stoichiometry of this nucleus, however, is not well established for Rad51, and values ranging from 2-3 (21) to 4-6 (19, 20) protein monomers were proposed.…”