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Article Dans Une Revue Nucleic Acids Research Année : 2007

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

Judith Miné
  • Fonction : Auteur
Ludovic Disseau
  • Fonction : Auteur
Giovanni Cappello
Marie Dutreix
  • Fonction : Auteur
Jean-Louis Viovy
  • Fonction : Auteur

Résumé

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.

Domaines

Biophysique
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Dates et versions

hal-00412889 , version 1 (02-09-2009)

Identifiants

  • HAL Id : hal-00412889 , version 1

Citer

Judith Miné, Ludovic Disseau, Masayuki Takahashi, Giovanni Cappello, Marie Dutreix, et al.. Real-time measurements of the nucleation, growth and dissociation of single Rad51–DNA nucleoprotein filaments. Nucleic Acids Research, 2007, 000 (000), pp.000. ⟨hal-00412889⟩
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