An Adenosine Triphosphate-dependent Deoxyribonuclease from Hemophilus influenzae Rd I. PURIFICATION AND PROPERTIES OF THE ENZYME

Abstract A deoxyribonuclease has been purified 2000-fold from Hemophilus influenzae Rd. The enzyme requires ATP and Mg2+ for the degradation of both double- and single-stranded DNA. The hydrolysis of single-stranded DNA proceeds at one-tenth the rate of duplex DNA. The enzyme degrades double-stranded DNA by producing large fragments which are then degraded further to oligonucleotides. It appears to degrade preferentially those molecules to which it has already become attached rather than moving randomly from one molecule to another for each bond scission. The limit product consists of 5'-phosphoryl, 3'-hydroxyl-terminated oligonucleotides, ranging in chain length from 1 to about 12 nucleotides. The average chain length of the limit product is 5.9, and the predominant species in the digest are di- and trinucleotides. The purified enzyme has a molecular weight of approximately 270,000 as determined by sucrose gradient sedimentation. However, ATP-dependent DNase activity has also appeared in a smaller species in some purified enzyme preparations, and two active components have been eluted from DEAE-cellulose and hydroxylapatite columns during the purification.