I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 21 



of the deoxyribonucleoside triphosphates of adenine, cytosine, or gua- 

 nine prevents DNA synthesis. Also, there is an absolute requirement for 

 Mg ions and DNA. Preincubation of the DNA with DNase (5 /j,g, 

 37°C, 30 minutes) completely destroyed its priming ability. In a similar 

 experiment, using a different P''--labeled nucleotide as an index of DNA 

 synthesis, it could be shown that dTTP is also required. 



2. Reverse Reaction 



Concentrations of inorganic pyrophosphate equal to that of the 

 individual triphosphates (1.6 X 10"^ M) have no effect on the synthetic 

 reaction rate. However, at pyrophosphate concentrations 200-fold higher 

 (3 X 10"'' M) the reaction rate is inhibited 50% and pyrophosphate is 

 incorporated into deoxyribonucleoside triphosphates, as shown in Table 

 VII. Unlike the synthetic reaction the reversal proceeds well in the 

 absence of three of the four triphosphates. Omission of all four, however, 

 reduces the rate of pyrophosphorolysis considerably. 



TABLE VII 

 Evidence for Reversal of Reaction" 



P*2_(Jeoxynucleoside 

 triphosphate'' 

 Sj'stem (m/xmoles) 



Complete system 3 . 47 



Omit DNA <0.02 



Omit enzyme <0.02 



OmitdATP 2.34 



Omit d ATP, dTTP 2.14 



Omit dATP, dTTP, dGTP 1 . 34 



Omit dATP, dTTP, dGTP, dCTP . 20 



Replace P^^P^z with Pi32 <0 . 02 



Replace DNA with DNase-treated DNA <0.02 



" From Bessman et al. (1958a). 



* These values are corrected for a zero-time value of 0.05 myumole. The complete sys- 

 tem contained 5 mMmoles each of dATP, dTTP, dGTP, and dCTP, 1 jumole of P'^pa 

 (5 X 10^ cpm /miole), 10 ng of DNA, 2 Mmole of MgCl2, 20 /imoles of tris buffer, pH 7.5, 

 and 0.8 fig of "polymerase" fraction VII in a final volume of 0.3 ml. After incubation 

 at 37°C for 30 minutes the mixture was chilled and cold solutions of albumin (0.05 ml 

 of 10 mg per ml), perchloric acid (0.25 ml of 1 A^), and "carrier" PP (0.5 ml of 0.1 M, 

 pH 7) were added. The nucleotides were adsorbed on charcoal which was then washed, 

 plated, and counted. 



That pyrophosphate is actually a product of the reaction is shown 

 in Table VIII. For each mole of dTMP incorporated into DNA, one mole 

 of pyrophosphate is released. The stoichiometry of this reaction and the 



