Separation and Characterization of Deoxyribonucleases of Escherichia coli B

Abstract

Abstract The soluble protein of Escherichia coli B was separated into six approximately equal fractions by precipitation with ammonium sulfate. These six fractions were further fractionated by chromatography with diethylaminoethyl cellulose. The chromatographic effluents were assayed for deoxyribonuclease activity with native deoxyribonucleic acid, heat-denatured DNA, and deoxyribo-oligonucleotides as substrates. This fractionation procedure separated five chromatographically distinct deoxyribonucleases from E. coli. Three of the enzymes have been previously described and were identified by their published properties. Endonuclease I was identified by its marked inhibition in the presence of soluble RNA, exonuclease I by its ability to degrade heat-denatured DNA, and exonuclease II by the DNA polymerase activity with which it is associated. Two distinct fractions with deoxyribo-oligonucleotidase activity were also separated from E. coli extract. These two oligonucleotidases, which we have termed exonuclease IV-A and exonuclease IV-B, are distinguished from the nucleases previously described by their ability to catalyze the release of acid-soluble products from oligonucleotides 20 times more rapidly than from either native or heat-denatured DNA. The two oligonucleotidases also cleave dinucleotides. Both enzymes produce 5'-mononucleotide products and are optimally active in alkaline solution. Although similar with respect to enzymatic properties, exonucleases IV-A and IV-B separate widely on diethylaminoethyl cellulose chromatography and rechromatography of either enzyme under a variety of conditions gives a single peak of enzymatic activity.