Abstract
SUMMARY Ionic strength inhibition of calf thymus polymerase Each tube contained the following concentrations: deoxyribo- nucleoside triphosphates, 40 pM in each (dGTP, 1060 c.p.m./ mpmole); Mg++, 8 mm; potassium phosphate, pH 7, 40 mM; mer- captoethanol, 1 InM; 82 rg of DNA (salmon sperm) ; and 120 pg of Fraction D protein. Final volume, 0.25 ml. All tubes were in- cubated 30 minutes at 35” and aliquots were assayed by the paper disk procedure (4). Molarities are the final concentration of added salt with the exception of the last entry, where molarity is total phosphate. The 0.04 M phosphate reaction, the control for all reactions in this series, incorporated 2.0 mpmoles of dGMPs2. Partial purification of a deoxyribonucleic acid (DNA)-synthe- sizing enzyme from calf thymus gland has been achieved. The purified enzyme preparation requires the deoxyribonucleoside triphosphates of adenine, guanine, cytosine, and thymine, Mg++, and primer DNA for activity. The synthetic reaction is in- hibited by pyrophosphate and, if radioactive pyrophosphate is used, it is incorporated into deoxyribonucleoside triphosphate. The over-all reaction may therefore be written: Deoxyribonucleoside triphosphates , DNA, Mg++
Highlights
MethodsAnalytical Methods-Protein was determined by the biuret reagent of Gornall et al [5]
The three purified polymerases have been isolated from quite different sources: bacteria exhibiting unlimited growth (l), regenerating liver in which the enzymes required for deoxyribonucleic acid (DNA) synthesis must be activated [2], and calf thymus in which a steady state of cell production is maintained
In all cases where primer was formed, the product synthesized by calf thymus polymerase in the presence of that primer DNA had a base incorporation ratio of the parent DNA preparation [21]
Summary
Analytical Methods-Protein was determined by the biuret reagent of Gornall et al [5]. Inorganic phosphate was determined by the method of Fiske and SubbaRow [6]. Pyrophosphate was determined as orthophosphate after 15 minutes’ hydrolysis in 1 N HzS04. Deoxyribose was determined with Dische’s diphenylamine reagent [7] or the indole reaction as described by Ceriotti [8]. Nucleotides were estimated by ultraviolet absorption, using the extinction coefficients of Volkin and Cohn [9]. Tritium was counted in windowless flow counters as described previously [10]
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