We have investigated quantitative molecular aspects of the interaction of recA protein with single-stranded DNA, by using a fluorescent modified-DNA referred to as etheno-M13 DNA. In addition, the effects of the nucleotide cofactors ATP and ADP, and the analogues ATP-γ-S, AMP-P-C-P, and AMP-P-N-P on this interaction have been studied. It is shown that ATP, AMP-P-N-P and, in particular. ATP-γ-S significantly increase the affinity of recA protein for single-stranded DNA, whereas ADP and, to a lesser degree, AMP-P-C-P decrease the affinity. Binding to etheno-M13 single-stranded DNA is co-operative, with the value of the co-operativity parameter, ω, being ≈50 under all conditions measured. The effect that ADP has on recA protein-DNA affinity is to lower the intrinsic binding constant, but it has no effect on the co-operativity of binding. In addition, the stability of the recA protein-DNA complex is very salt dependent ( d log K d log [Nacl] ≈ -10) and it is the intrinsic binding affinity rather than the co-operativity of binding that is affected: Thus, under all conditions observed, recA protein binds single-stranded DNA co-operatively with a value of ω = 50 ± 10. The binding affinity is also influenced by the type of anion present, being ≈10,000-fold higher when acetate ion is present instead of chloride ion. These data have been interpreted to suggest that recA protein forms up to five ionic interactions when it binds to single-stranded DNA and that five to six anions are displaced upon binding. The modulation of recA protein-DNA complex stability by nucleotide cofactors suggests that these cofactors play a role in the cycling of recA protein on and off single-stranded DNA, with ATP being required for DNA binding under physiological conditions and ADP serving as a "release" factor. These results are discussed in terms of a model for the role of ATP hydrolysis in a recA protein-single-stranded DNA binding cycle.
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