Differential reactivities at restriction enzyme sites

Abstract

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and ΦX174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in ΦX174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than does the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.