Direct observation of individual RecA filaments assembling on single DNA molecules
- 20 September 2006
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature
- Vol. 443 (7113), 875-878
- https://doi.org/10.1038/nature05197
Abstract
Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination1. Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth2,3,4,5,6,7. However, many aspects of this process remain unclear, including the rates of nucleation and growth and the involvement of ATP hydrolysis, largely because visualization at the single-filament level is lacking. Here we report the direct observation of filament assembly on individual double-stranded DNA molecules using fluorescently modified RecA. The nucleoprotein filaments saturate the DNA and extend it ∼1.6-fold. At early time points, discrete RecA clusters are seen, permitting analysis of single-filament growth from individual nuclei. Formation of nascent RecA filaments is independent of ATP hydrolysis but is dependent on the type of nucleotide cofactor and the RecA concentration, suggesting that nucleation involves binding of ∼4–5 ATP–RecA monomers to DNA. Individual RecA filaments grow at rates of 3–10 nm s-1. Growth is bidirectional and, in contrast to nucleation, independent of nucleotide cofactor, suggesting addition of ∼2–7 monomers s-1. These results are in accord with extensive genetic and biochemical studies, and indicate that assembly in vivo is controlled at the nucleation step. We anticipate that our approach and conclusions can be extended to the related eukaryotic counterpart, Rad51 (see ref.8), and to regulation by assembly mediators9,10,11.Keywords
This publication has 23 references indexed in Scilit:
- The RecA Binding Locus of RecBCD Is a General Domain for Recruitment of DNA Strand Exchange ProteinsMolecular Cell, 2006
- Catalyst of a catalystNature, 2005
- Rad51 Recombinase and Recombination MediatorsJournal of Biological Chemistry, 2003
- Polymerization and mechanical properties of single RecA–DNA filamentsProceedings of the National Academy of Sciences of the United States of America, 1999
- The Translocating RecBCD Enzyme Stimulates Recombination by Directing RecA Protein onto ssDNA in a χ-Regulated MannerCell, 1997
- HOMOLOGOUS PAIRING AND DNA STRAND-EXCHANGE PROTEINSAnnual Review of Biochemistry, 1994
- Biochemistry of Genetic Recombination: Energetics and Mechanism of DNA Strand ExchangeAnnual Review of Biophysics and Biophysical Chemistry, 1991
- Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA proteinBiochemistry, 1988
- Structure of helical RecA-DNA complexes: Complexes formed in the presence of ATP-gamma-S or ATPJournal of Molecular Biology, 1986
- Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregationJournal of Molecular Biology, 1977