Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE
Novikoff hepatoma has been 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 molecular weight of 32 000 as determined by
sodium dodecylsulfate-
polyacrylamide gel electrophoresis. In
sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the
hydroxylapatite fraction
enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent
cation for maximal activity. The apparent Km for total deoxyribonucleoside
triphosphate is 7-8 muM and for
DNA 125 mug/ml. Activated
DNA, rendered 7%
acid soluble by
DNase I, is the preferred primer-template, although a number of synthetic
polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ 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 sstimulated about twofold 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.