An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII.

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.