Abstract
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.
Highlights
In the presence of ATP, the found t o be influenced by NaCl and ADP concentration.An transfer reaction is dependent on the concentrationof increase in either the salt or ADP concentration resulted in the competitor single-stranded DNA, poly(dT)
Nucleotides-The transfer reaction was conducted in the pres- 15 to 55 p~ (Fig. lA).The rate of the slow component is ence of 250 p~ ATP to determine the affect of this cofactor relatively unaffected up to 35 FM poly(dT), but thendecreases on the transerate. recA protein-etheno M13 DNA complexes by almost %fold, as poly(dT) concentration isincreased furwere formed, excess poly(dT) was added to initiate the ther (Fig. 1B)
Because ATP binding and hydrolysis stimulates the renaturation reaction (McEntee, 1985) and are required during the strand exchange reaction (Cox and Lehman, 1981), we have analyzed the effect of ATP on the transfer reaction
Summary
A part of the DNA is influenced by the presence of NaCl, ADP, and ATP mechanism involves the formationof a kinetic ternary (Menetski and Kowalczykowski, 1985a). In the presence of ATP, the found t o be influenced by NaCl and ADP concentration.An transfer reaction is dependent on the concentrationof increase in either the salt or ADP concentration resulted in the competitor single-stranded DNA, poly(dT). This an increase in thoebserved rate of transfer. Some of this data was presented in abstract form at the 190th Annual American Chemical Society Meeting in Chicago (Menetski and Kowalczykowski, 198513) and the
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