Journal
NUCLEIC ACIDS RESEARCH
Volume 37, Issue 12, Pages 4089-4099Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkp326
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Funding
- Dutch organization for Fundamental Research of Matter (FOM)
- Netherlands Organization for Scientific Research (NWO)
- Netherlands Genomics Initiative/NWO
- European Commission
- Integrated Projects Molecular Imaging and DNA Repair [512113]
- National Cancer Institute-NIH USA
- The Netherlands Organization for Scientific Research
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RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA-ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA-ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.
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