Real-time measurements of the nucleation, growth and dissociation of single Rad51-DNA nucleoprotein filaments.
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.
Miné
Judith
J
Laboratoire Physico-Chimie Curie, UMR CNRS 168, Institut Curie, Paris, France.
Disseau
Ludovic
L
Takahashi
Masayuki
M
Cappello
Giovanni
G
Dutreix
Marie
M
Viovy
Jean-Louis
JL
D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001704:Biopolymers / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D052898:Optical Tweezers; D051135:Rad51 Recombinase / Q000378:metabolism*
D000227:Adenine Nucleotides; D001704:Biopolymers; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase