Transfer of recA protein from one polynucleotide to another. Effect of ATP and determination of the processivity of ATP hydrolysis during transfer.
The transfer of recA protein from a fluorescently modified single-stranded DNA, containing 1,N6-ethenoadenosine and 3,N4-ethenocytosine, to polydeoxythymidylic acid (poly(dT)) was shown to occur by a complex mechanism in both the absence and presence of ADP (Menetski, J. P., and Kowalczykowski, S. C. (1987) J. Biol. Chem. 262, 2085-2092). A part of the mechanism involves the formation of a kinetic ternary intermediate. Since the binding and hydrolysis of ATP by recA protein is involved in many of the recA protein in vitro activities, we have analyzed the effect of ATP on the transfer reaction. In the presence of ATP, the transfer reaction is dependent on the concentration of the competitor single-stranded DNA, poly(dT). This result suggests that transfer does not occur by a simple dissociation mechanism. The reaction occurs via two kinetically distinct species of protein X DNA complexes with properties that are similar to those characterized for the transfer reaction in the absence of ATP. There is a complicated effect of nucleotide concentration on the rate of transfer. At low concentrations of ATP (less than 50 microM), increasing nucleotide concentration increases the rate of transfer; this is similar to the effect of ADP. However, at high concentrations of ATP (greater than 50 microM), increasing ATP concentration decreases the rate of transfer. Finally, the processivity of ATP hydrolysis during transfer was found to increase with increases in ATP concentration. Less than one ATP molecule was hydrolyzed per transfer event at low ATP concentrations (less than 20 microM) while greater than 50 molecules were hydrolyzed at high ATP concentration (greater than 250 microM). These data suggest that the rate of transfer is not directly coupled to the rate of hydrolysis.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001693:Biological Transport, Active / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011071:Poly T / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*
D015636:Magnesium Chloride; D011071:Poly T; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D008274:Magnesium