Publication Date:
2012-10-30
Description:
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 DNA. 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 assembly. 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 attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. 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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112059/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112059/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bell, Jason C -- Plank, Jody L -- Dombrowski, Christopher C -- Kowalczykowski, Stephen C -- CA136103/CA/NCI NIH HHS/ -- F32 CA136103/CA/NCI NIH HHS/ -- GM62653/GM/NIGMS NIH HHS/ -- GM64745/GM/NIGMS NIH HHS/ -- R01 GM062653/GM/NIGMS NIH HHS/ -- R01 GM064745/GM/NIGMS NIH HHS/ -- T32 CA10052159/CA/NCI NIH HHS/ -- T32 CA108459/CA/NCI NIH HHS/ -- T32 GM007377/GM/NIGMS NIH HHS/ -- England -- Nature. 2012 Nov 8;491(7423):274-8. doi: 10.1038/nature11598. Epub 2012 Oct 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Microbiology, University of California, Davis, California 95616, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23103864" target="_blank"〉PubMed〈/a〉
Keywords:
DNA, Single-Stranded/chemistry/*metabolism
;
DNA-Binding Proteins/*metabolism
;
Escherichia coli/*chemistry/enzymology
;
Escherichia coli Proteins/*metabolism
;
Hydrogen-Ion Concentration
;
Ligands
;
Microscopy, Fluorescence/*methods
;
Models, Biological
;
Models, Molecular
;
Molecular Conformation
;
Protein Multimerization
;
Rec A Recombinases/*chemistry/*metabolism
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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