Direct observation of individual RecA filaments assembling on single DNA molecules.
Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination. 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 growth. 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 approximately 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 approximately 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 approximately 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.), and to regulation by assembly mediators.
Galletto
Roberto
R
Section of Microbiology, Center for Genetics and Development, Davis, California 95616, USA.
Amitani
Ichiro
I
Baskin
Ronald J
RJ
Kowalczykowski
Stephen C
SC
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D006868:Hydrolysis; D020836:Protein Structure, Quaternary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism*
D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases