Stable binding of recA protein to duplex DNA. Unraveling a paradox.
recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D010957:Plasmids; D011938:Rec A Recombinases / Q000378:metabolism*
D004270:DNA, Circular; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases