Nucleic Acids Research Advance Access originally published online on August 20, 2007
Nucleic Acids Research 2007 35(17):5646-5657; doi:10.1093/nar/gkm629
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Nucleic Acids Research, 2007, Vol. 35, No. 17 5646-5657
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules
1Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, 2Department of Cell Biology and Genetics and 3Department of Radiation Oncology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands
*To whom correspondence should be addressed. Tel: +31 15 2786094, Fax: +31 15 2781202, Email: c.wyman{at}erasmusmc.nl, c.dekker{at}tudelft.nl
Received May 16, 2007. Revised July 24, 2007. Accepted July 31, 2007.
The human DNA repair protein RAD51 is the crucial component of helical nucleoprotein filaments that drive homologous recombination. The molecular mechanistic details of how this structure facilitates the requisite DNA strand rearrangements are not known but must involve dynamic interactions between RAD51 and DNA. Here, we report the real-time kinetics of human RAD51 filament assembly and disassembly on individual molecules of both single- and double-stranded DNA, as measured using magnetic tweezers. The relative rates of nucleation and filament extension are such that the observed filament formation consists of multiple nucleation events that are in competition with each other. For varying concentration of RAD51, a Hill coefficient of 4.3 ± 0.5 is obtained for both nucleation and filament extension, indicating binding to dsDNA with a binding unit consisting of multiple (
4) RAD51 monomers. We report Monte Carlo simulations that fit the (dis)assembly data very well. The results show that, surprisingly, human RAD51 does not form long continuous filaments on DNA. Instead each nucleoprotein filament consists of a string of many small filament patches that are only a few tens of monomers long. The high flexibility and dynamic nature of this arrangement is likely to facilitate strand exchange.
Present address: Ralf Seidel, Biotechnological Center, Dresden University of Technology, Tatzberg 47-51, 01307 Dresden, Germany.
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