Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

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Abstract

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA1. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly2,3. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts2,3,4,5. Previous single-molecule assays have measured the nucleation and growth of RecA—and its eukaryotic homologue RAD51—on naked double-stranded DNA and ssDNA6,7,8,9,10,11,12; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA3. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

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Figure 1: Direct visualization of RecA filament assembly on single molecules of SSB-coated ssDNA shows that RecA nucleates as a dimer.
Figure 2: RecA filaments grow via rapid addition of monomers.
Figure 3: RecA filament growth on SSB-coated ssDNA is bidirectional.
Figure 4: Nucleation is modulated by ligand binding, repressed at physiological pH, and enhanced by RecOR and RecFOR; growth is enhanced by RecOR.

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Acknowledgements

We are grateful to members of the laboratory for their comments on this work. J.C.B. was funded by a National Institutes of Health (NIH) Predoctoral Training Program in Molecular & Cellular Biology (T32 GM007377); C.C.D. and J.L.P. were funded by the NIH T32 Training Program in Oncogenic Signals and Chromosome Biology (CA10052159); J.L.P. was also funded by a NIH Postdoctoral Fellowship (CA136103); and S.C.K. was supported by the NIH (GM62653 and GM64745).

Author information

J.C.B., J.L.P., C.C.D. and S.C.K. conceived the general ideas, designed the experiments and interpreted the data. J.C.B., J.L.P. and C.C.D. performed experiments. J.C.B. and S.C.K. wrote the manuscript.

Correspondence to Stephen C. Kowalczykowski.

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Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-6 and full legends for Supplementary Videos 1 and 2. (PDF 2723 kb)

Direct imaging of nucleation and growth of RecA on SSB-coated ssDNA

Video illustrating the procedure and showing direct imaging of nucleation and growth of RecA on SSB-coated ssDNA. See Supplementary Information file for full legend. (MOV 3132 kb)

Optical trapping and manipulation of single molecules of gapped DNA for direct imaging of RecAf filament assembly

Video illustrating the procedure and showing optical trapping and manipulation of single molecules of gapped DNA for direct imaging of RecAf filament assembly. See Supplementary Information file for full legend. (MOV 9278 kb)

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Bell, J., Plank, J., Dombrowski, C. et al. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491, 274–278 (2012) doi:10.1038/nature11598

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