Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules.
Rad54 protein plays an important role in the recombinational repair of double-strand DNA (dsDNA) breaks. It is a dsDNA-dependent ATPase that belongs to the Swi2/Snf2 family of chromatin-remodeling proteins. Rad54 remodels (1) DNA structure, (2) chromatin structure, and (3) Rad51-dsDNA complexes. These abilities imply that Rad54 moves along DNA. Here, we provide direct evidence of Rad54 translocation by visualizing its movement along single molecules of dsDNA. When compared to the remodeling processes, translocation is unexpectedly rapid, occurring at 301 +/- 22 bp/s at 25 degrees C. Rad54 binds randomly along the dsDNA and moves in either of the two possible directions with a velocity dependent on ATP concentration (K(m) = 97 +/- 28 microM). Movement is also surprisingly processive: the average distance traveled is approximately 11,500 bp, with molecules traversing up to 32,000 bp before stopping. The mechanistic implications of this vigorous Rad54 translocase activity in chromatin and protein-DNA complex remodeling are discussed.
Amitani
Ichiro
I
Section of Microbiology, University of California, 95616, USA.
Baskin
Ronald J
RJ
Kowalczykowski
Stephen C
SC
D000255:Adenosine Triphosphate / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003952:Diagnostic Imaging; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D021381:Protein Transport; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
D002843:Chromatin; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D016650:Fluorescein-5-isothiocyanate