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Direct observation of individual RecA filaments assembling on single DNA molecules

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.

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Figure 1: Nucleation and growth of RecA filaments visualized on individual dsDNA molecules.
Figure 2: Nucleation of RecA filaments is independent of ATP hydrolysis but dependent on the type of nucleoside triphosphate and the RecA concentration.
Figure 3: Growth of individual RecA filaments is independent of nucleoside triphosphate.
Figure 4: RecA filaments can grow bidirectionally in the presence of ATP.

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Acknowledgements

We thank M. Spies, J. Siino and A. Forget for suggestions and discussions, and members of the Kowalczykowski laboratory for comments. This work was supported by NIH grants to S.C.K. and R.J.B. R.G. was supported partially by a Fellowship from the Jeane B. Kempner Foundation.

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Correspondence to Stephen C. Kowalczykowski.

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Supplementary Notes

This file contains Supplementary Figures 1–13, Supplementary Methods and related text. Supplementary Figure 1 is a simple schematic that summarizes the main finding of the paper. The Supplementary Methods describe data analysis and preparation of fluorescent RecA; Supplementary Data provide biochemical characterization the fluorescent RecA. The data included show RecA–ATPγS filaments are highly stable; nucleation becomes rate-limiting for RecA assembly at high NaCl concentrations; RecA preferentially nucleates at AT-rich DNA; the relationship between DNA elongation and RecA filament formation; and stabilization of ATP–RecA clusters by Ca2+ ions. (PDF 471 kb)

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Galletto, R., Amitani, I., Baskin, R. et al. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006). https://doi.org/10.1038/nature05197

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