I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 23 



" From Bessman et al. (1958a). 



* The labeled substrate was dTP32p32P-2-C". 



' dATP was omitted. 



requirements for the system have led to the following equation for the 

 synthesis of DNA (Romberg, 1960) : 



+ 4 (n) PP 



The mechanism of the reaction is viewed by Koraberg (1960) as the 

 nucleophilic attack of the terminal 3'-hydroxyl group of the DNA chain 

 on the nucleotidyl phosphorus atom of the deoxyribonucleoside triphos- 

 phate with the elimination of pyrophosphate. By this mechanism, the 

 DNA chain is lengthened from the nucleoside and as depicted in Fig. 6. 

 This mechanism is consistent with all the present data. 



C. THE LIMITED REACTION (Adler et al., 1958) 



In the previous section (IV,B,1), a requirement for all four deoxy- 

 ribonucleoside triphosphates was shown to be necessaiy for DNA syn- 

 thesis. However, using highly labeled substrates, a small but significant 

 incorporation of single deoxyribonucleotides can be demonstrated (Table 

 IX). This incorporation amounts to approximately 0.1% of the rate of 

 the complete system and occurs with any of the four deoxyribonucleoside 

 triphosphates. The reaction proceeds rapidly at first and then falls off, 

 suggesting that there are preferred sites of attachment which become 

 saturated. Several lines of evidence suggest that this reaction is not due 

 to the presence of small amounts of the other deoxyribonucleotides in 

 the enzyme preparation, in the added DNA, or in the labeled triphos- 

 phate; and the possibility that the other triphosphates are formed by 

 pyrophosphorolysis of the DNA during the incubation has been ruled out. 



When a product of this limited reaction (containing a labeled deoxy- 

 ribonucleotide) is isolated, washed free of acid-soluble deoxyribonucleo- 

 tides and hydrolyzed with snake venom phosphodiesterase, almost all 



