I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 33 



TABLE XV 



Effect of Relative Substrate Concentrations 

 ON Composition of Synthetic DNA" 



Products 



Substrates (relative molar concentration) 



Net A + T A + G 



dCTP dGTP dTTP dATP synthesis G + C C + T 



1.0 1.0 1.0 1.0 IIX 1.98 1.00 



1.0 1.0 0.2 1.0 6X 1.82 1.04 



1.0 1.0 0.2 0.2 6X 1.82 0.97 



T2 bacteriophage DNA as primer 1 . 92 . 98 



" From Lehman et al. (1958a). 



b. Degradation of each of the products in such a way that the P^^ 

 introduced into the DNA in the form of the specific deoxyribonucleotide 

 is now part of the particular deoxyribonucleotide to which the labeled 

 triphosphate was attached. This is accomplished by incubating the indi- 

 vidual products with micrococcal DNase (Cunningham et at., 1956), and 

 splenic phosphodiesterase (Heppel et al., 1953; Hilmoe, 1960). 



c. Separation of the deoxyribonucleotides and estimation of their P^- 

 content. This is accomplished by paper electrophoresis (]\larkham and 

 Smith, 1953). 



For a detailed description of this technique, the reader is referred 

 to the original paper of Josse et al. (1961). A brief description of a 

 typical determination follows: Four different reaction mixtures con- 

 taining one labeled and three unlabeled triphosphates are incubated and 

 the reaction is terminated by the addition of carrier DNA and perchloric 

 acid. Let us consider the product containing dAMP^-. If we examine 

 Fig. 9 we see that dATP^- (designated as Y-nucleotide) adds to the 

 growing deoxyribonucleotide chain by forming a 3'-5'-phosphodiester 

 bridge with the terminal deoxynucleotide (designated X-nucleotide). This 

 will occur at specific points in the newly synthesized DNA and these 

 positions are presumably dictated by base-pairing with the template 

 DNA. The product is washed free of acid-soluble unreacted deoxy- 

 ribonucleoside triphosphates and degraded enzymatically as shown in 

 Fig. 9. Using the same designation as for synthesis, it can be seen that 

 since the enzymes specifically hydrolyze the phosphodiester bridges 

 between the 5'-carbon and the phosphorus, the P^^ from the incorporated 

 dAMP^- (Y) now ends up on the 3'-position of the neighboring deoxy- 

 ribonucleotide (X). These 3'-deoxyribonucleotides are separated and 

 analyzed for P^^ content and the fraction of each of the four possible 

 dinucleotide pairs containing dAMP as the 5'-deoxyribonucleotide can 



