Uptake of homologous single-stranded fragments by superhelical DNA: II. Characterization of the reaction

We have studied the association of superhelical DNA (RFI)³ of phage G4 with defined single-stranded fragments isolated after cleavage of viral (+) strands by endonuclease R · HaeIII. The sedimentation rates of complexes formed by uptake of different single-stranded restriction fragments by G4 RFI were consistent with the view that base-pairing between the two components causes unwinding of superhelical turns, with one negative superhelical turn removed for every ten nucleotide residues of third strand taken up. The combining ratio of superhelical DNA and a single specific fragment was close to unity.At high concentrations of salt, nitrocellulose filters efficiently retained complexes of superhelical DNA and homologous fragments, which provided the basis for a rapid assay, and permitted the estimation of the thermodynamic and kinetic parameters of strand uptake in vitro. The reaction is reversible, with an apparent K_eq of approximately 10⁶m^?1. Apparent rate constants, k₁, for uptake of different fragments (85 to 1100 nucleotides long) varied about fourfold, with no obvious relationship to the length of the fragment. In 10 mm-Tris · HCl (pH 7.5), 200 mm-NaCl, fragments were taken up most rapidly at about 75 °C. Under these conditions, the apparent k₁ for a fragment 250 nucleotides long was approximately 600 m^?1s^?1, which is two or three orders of magnitude slower than the calculated rate of association of complementary strands of that length. At physiological temperatures, appreciable rates of strand uptake were seen only at low concentrations of salt (4 mm-Na⁺ in 10 mm-Tris · HCl), and were one or two orders of magnitude less than the rate at 75 °C in 200 mm-NaCl. At a given concentration of counterion a threshold temperature exists above which the rate of reaction rises sharply from an undetectable level.Thermodynamic calculations indicate that the reaction is entropically driven, and that the rate is limited by a step exhibiting a positive entropy and enthalpy of activation. The data are consistent with a model for strand uptake in which the rate-limiting step is the unstacking of a small number of base-pairs in the superhelical DNA. Stabilization and extension of the nucleus of base-pairs formed with the incoming strand is favored by the decrease in free energy associated with removal of superhelical turns.