Quantitative RecA protein binding to the hybrid duplex product of DNA strand exchange.
Following a DNA strand exchange reaction, RecA protein remains bound to the hybrid DNA product. DNA strand exchange reactions were carried out under optimal conditions in the presence of both RecA protein and SSB protein. As monitored by a sensitive DNA underwinding assay, all of the RecA protein present in the RecA nucleoprotein filament that initiates the strand exchange reaction can be accounted for on the hybrid DNA. As shown elsewhere, the SSB is bound to the displaced single DNA strand. Previous studies showed that RecA protein will dissociate from dsDNA when ADP levels build up, or transfer from dsDNA to ssDNA when the latter is not bound by SSB. The present work (done with ATP regeneration and SSB) shows that efficient strand exchange occurs in the absence of a net dissociation or transfer of RecA monomers from the filament. Such a dissociation or transfer is therefore not a mechanistic requirement for DNA strand exchange. The results provide evidence against some models proposed for the DNA strand exchange mechanism.
Ullsperger
C J
CJ
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Cox
M M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases