19] Preparation and properties of the HpaI and HpaII endonucleases

Abstract

Publisher Summary This chapter discusses the preparation and properties of the HpaI and HpaII endonucleases. Two restriction endonuclease activities, HpaI and HpaII are isolated from Haemophilus parainfluenzae. Endonuclease HpaI has been isolated in homogeneous form, while HpaII has been purified free of contaminating nuclease and phosphatase activities. HpaI recognizes the DNA sequence and cleaves the phosphodiester bonds in both strands as indicated. HpaII recognizes the DNA sequence and cleaves the phosphodiester bonds in a staggered fashion. Assay method is further explained. Two assays have been used during the purification of HpaI and HpaII. The first assay is qualitative and involves the cleavage of γ DNA by the respective enzyme into fragments of discrete size. These fragments are separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Characteristic sets of fragments are seen for each of the enzymes. HpaI cleaves γ DNA 11 times while HpaII cleaves γ, DNA more than 50 times. The second assay is quantitative and measures the conversion of closed circular RF fd DNA, which contains a single HpaI site, to the linear form which is sensitive to degradation by RecBC DNase.