Organized unidirectional waves of ATP hydrolysis within a RecA filament.
The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 degrees C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k(cat) of 20.8 +/- 1.5 min(-1). Under the same conditions, the rate of end-dependent filament disassembly (k(off)) is 123 +/- 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k(cat) to k(off) infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.
Cox
Julia M
JM
Department of Biochemistry, University of Wisconsin, Madison, USA.
Tsodikov
Oleg V
OV
Cox
Michael M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D011938:Rec A Recombinases / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases
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