RAPID-REACTION ANALYSIS OF PLASMID DNA CLEAVAGE BY THE ECORV RESTRICTION-ENDONUCLEASE

Rapid-reaction methods have been used previously to identify intermedi ates in the reaction of the EcoRV restriction endonuclease on oligonuc leotide substrates. In this study, the pathway on macromolecular DNA w as elucidated by using the quench-flow method to analyze EcoRV reactio ns on a plasmid with one recognition site. Some reactions were carried out by first allowing the EcoRV enzyme to bind nonspecifically to the DNA and then initiating DNA cleavage by adding magnesium ions. The su bsequent transfer of the enzyme from nonspecific to specific sites was extremely rapid, at a random walk rate of at least 5 x 10(5) base pai rs per second. The two strands of the DNA at the EcoRV recognition sit e were then cleaved sequentially, at rates that were faster than the t urnover number of the enzyme. The rates recorded for the cleavage step s were direct measurements of phosphodiester hydrolysis, while the tur nover is limited by the dissociation of the product cleaved in both st rands. Other reactions were initiated by adding EcoRV and MgCl2 to the DNA: these revealed not only the processes observed in reactions star ting from DNA-bound enzyme but also the bimolecular association of the protein with the plasmid. The association rate was limited by diffusi on but its rate constant, 1.2 x 10(8) M-1 s(-1), was unusually small f or the binding of a protein to DNA. The slowness of this diffusion-con trolled process may be due to a rapid oscillation of the protein betwe en closed and open conformations, with only the open form capable of b inding DNA.