Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous β polymerase

Abstract

DNA polymerase-.beta. (EC 2.7.7.7) from the Novikoff hepatoma was purified over 200,000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60,000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a MW of 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the MW is estimated at 31,000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff .beta.-polymerase requires all 4 deoxyribonucleoside triphosphates, primer-template and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 .mu.M and for DNA 125 .mu.g/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can be efficiently utilized, particularly in the presence of Mn2+. The Mg2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The .beta.-polymerase is stimulated about 2-fold by NaCl or KCl at an optimum of 50-100 mM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most .beta.-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.