Published online 8 June 2005
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Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function
1Department of Cell Biology and Genetics, Erasmus Medical Center PO Box 1738, 3000 DR Rotterdam, The Netherlands 2Kavli Institute of Nanoscience, Delft University of Technology Lorentzweg 1, 2628 CJ Delft, The Netherlands 3Department of Radiation Oncology, Erasmus Medical Center-Daniel Rotterdam, The Netherlands
*To whom correspondence should be addressed. Tel: +31 10 408 8337; Fax: +31 10 408 9468; Email: c.wyman{at}erasmusmc.nl
Received April 22, 2005. Accepted May 19, 2005.
Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.
Present address: John van Noort, Department of Biophysics, Huygens Laboratory, Leiden University, 2300 RA Leiden, The Netherlands
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