Supercoiling and the mechanism of restriction endonucleases

We have used topoisomerase I in the presence of netropsin and ethidium bromide to generate DNA molecules of varying superhelical density. Digestion by endonuclease EcoRI is sensitive to supercoiling, being maximal for the relaxed form. Endonucleases AvaI and BamHI, by contrast, are relatively unaffected. The results are interpreted in terms of the base composition of the DNA in the vicinity of these sites. dA + dT-rich regions are more susceptible to deformation than are dG + dC-rich ones. Analysis of the rates of disappearance of linear molecules confirms a two-step mechanism for EcoRI cleavage but suggests that BamHI and AvaI cleave both strands simultaneously.