Communications between distant sites on supercoiled DNA from non-exponential kinetics for DNA synapsis by resolvase.
To determine how distant sites on supercoiled DNA communicate with each other, the mechanism of site-specific recombination by resolvase was analysed by using a rapid-reaction quench-flow device to study the kinetics of individual steps in the reaction pathway. Three sets of measurements revealed the rates for: (1) the initial binding of the protein to its target sites on the DNA; (2) the synapsis of the two DNA-protein complexes; (3) the overall process of recombination. The binding of the protein to the DNA was complete within 50 milliseconds while recombination required 500 seconds. Surprisingly, synapsis spanned this entire time range: some DNA molecules gave synaptic complexes within ten milliseconds after the initial binding, while others took over 100 seconds. The departure from exponential behaviour may be due to each molecule 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 current 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. Alternatively, the non-exponential kinetics for synapsis may be due to multiple re-arrangements of non-productive complexes following DNA juxtaposition.
Oram
M
M
Department of Biochemistry, University of Bristol, UK.
Marko
J F
JF
Halford
S E
SE
D002021:Buffers; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D007700:Kinetics; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011995:Recombination, Genetic / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D019895:Transposases
D002021:Buffers; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D018698:Glutamic Acid; D012965:Sodium Chloride; D004254:DNA Nucleotidyltransferases; D019895:Transposases