Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA

Abstract

Two different methods have been used to locate the recognition sites for the restriction endonuclease from Escherichia coli K12 on the genome of bacteriophage PM2. The first method employs purified fragments of PM2 DNA produced by digestion with Hin dIII. The fragments are tested for their ability to support the ATPase activity of the enzyme or, alternatively, to inhibit it in the presence of intavt substrate DNA. In this way, recognition sites were assigned to the Hin dIII fragments 2, 6 and 7. An independent approach involved the study of complexes between the enzyme and either the Hin dIII fragments or linear DNA produced by cleavage of PM2 DNA at the unique site for Hpa II. Electron microscopic analysis of such preparations allowed mapping of the three recognition sites to 40, 60 and 62 map units from the Hpa II cleavage site.