Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.
An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.
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