RecA filament dynamics during DNA strand exchange reactions.
The role of ATP hydrolysis in RecA protein-mediated DNA strand exchange reactions remains controversial. Competing models suggest that ATP hydrolysis is coupled either to a simple redistribution of RecA monomers within a filament to repair filament discontinuities, or more directly to rotation of the DNA substrates to drive branch movement unidirectionally. Here, we test key predictions of the RecA redistribution idea. When ATP is hydrolyzed, DNA strand exchange is accompanied by a RecA exchange reaction, between free and bound RecA protomers in the interior of RecA filaments, that meets a central prediction of the model. The RecA protomer exchange is not required for, and does not occur during, the "search for homology" in which the single-stranded DNA within a RecA-ssDNA nucleoprotein filament is homologously aligned with the duplex DNA. Instead, the RecA exchange is triggered by the completion of strand exchange (a strand switch to generate a hybrid DNA product) in any given segment of the filament. In effect, formation of hybrid DNA leads to a change in filament conformation to one with properties approximating those of RecA filaments bound to double-stranded DNA. Addition of the RecA K72R mutant protein to a reaction with the wild type protein leads to the formation of mixed filaments and a poisoning of the DNA strand exchange reaction. Under some conditions, a facile RecA protomer exchange is observed, and significant ATP is hydrolyzed, even though DNA strand exchange is entirely blocked by the mutant protein. A redistribution of RecA protomers coupled to ATP hydrolysis is not sufficient in itself to explain how ATP hydrolysis facilitates DNA strand exchange. A RecA protomer exchange may nevertheless play an important role in the DNA strand exchange process.
Shan
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Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Cox
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D000255:Adenosine Triphosphate / Q000378:metabolism; D017104:Bacteriophage M13; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D006868:Hydrolysis; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D009698:Nucleoproteins / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009698:Nucleoproteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases