COMMUNICATIONS BETWEEN DISTANT SITES ON SUPERCOILED DNA FROM NONEXPONENTIAL KINETICS FOR DNA SYNAPSIS BY RESOLVASE

To determine how distant sites on supercoiled DNA communicate with eac h other, the mechanism of site-specific recombination by resolvase was analysed by using a rapid-reaction quench-flow device to study the ki netics of individual steps in the reaction pathway. Three sets of meas urements revealed the rates for: (1) the initial binding of the protei n to its target sites on the DNA; (2) the synapsis of the two DNA-prot ein complexes; (3) the overall process of recombination. The binding o f the protein to the DNA was complete within 50 milliseconds while rec ombination required 500 seconds. Surprisingly, synapsis spanned this e ntire time range: some DNA molecules gave synaptic complexes within te n milliseconds after the initial binding, while others took over 100 s econds. The departure from exponential behaviour may be due to each mo lecule of DNA having to undergo different conformational fluctuations in order to juxtapose the recombinational sites. From polymer physics theory, the rate of synapsis ought to vary with either the size of the DNA molecule or the length of DNA between the recombinational sites, depending on the nature of the fluctuations, but plasmids of different sizes and with different spacings between the sites all gave the same rates for synapsis. This observation cannot be reconciled with curren t models for encounters of distant sites on supercoiled DNA. However, the superhelical axis in the DNA molecules used here will be branched at one or more positions and the encounters may arise from the motion of a single branch relative to the remainder of the chain. Alternative ly, the non-exponential kinetics for synapsis may be due to multiple r e-arrangements of non-productive complexes following DNA juxtaposition . (C) 1997 Academic Press Limited.