140 H. GOBIND KHORANA 



was the same as that of cytosine. Hence, there was the characteristic equiva- 

 lence between the purine and pyrimidine content. Furthermore, the ratio of 

 adenine -f- thymine/guanine + cytosine was always close to that found for 

 the primer used. 



The same results were obtained when the net synthesis had proceeded 

 to furnish largely new product as well as at very early stages of the syn- 

 thetic process. Furthermore, the characteristics of the base composition 

 were maintained even when in the reaction mixtures the relative concen- 

 trations of the individual triphosphates were varied. 90 



The above results and the findings reported below on the incorporation 

 of analogs in the enzymically synthesized products 91 lead to the conclusion 

 that the selection of precursors according to their hydrogen bond forming 

 characteristics, is an important feature of the reaction catalyzed by the 

 polymerizing enzyme. Furthermore, the influence of the primer DNA on 

 the composition of the synthetic products provides support for the biologi- 

 cally attractive idea that the primer DNA serves as a template for the 

 synthesis of new DNA. 



e. The Incorporation of Purine and Pyrimidine Analogs 91 



Extensive studies in vivo have previously shown that certain structural 

 analogs of the naturally found purines and pyrimidines may be incorpo- 

 rated into DNA. Studies with the bacterial polymerase using the triphos- 

 phates of several of the familiar analogs have shown again that the enzymic 

 reaction is governed by the specific base-pairing rule in the Watson-Crick 

 model. In each case, the analog substitutes specifically for the base it closely 

 resembles with respect to its hydrogen-bonding properties. Thus, in the 

 complete reaction mixture, 5-bromodeoxyuridine-5'- as well as deoxyuri- 

 dine-5 '-triphosphates can replace thymidine-5 '-triphosphate but not any 

 one of the other three essential precursors (deoxy adenosine, deoxyguano- 

 sine, and deoxycytidine triphosphates). Similarly 5-bromo- and 5-methyl- 

 deoxycytidine-5'-triphosphates replaced only deoxycytidine triphosphate 

 in the full complement of the triphosphates. A purine analog, deoxy inosine 

 triphosphate could replace deoxyguanosine triphosphate but not any one 

 of the other triphosphates. It may be noted that the synthetic rates in the 

 presence of the various analogs varied considerably. 



In the case of the synthetic product obtained when labeled deoxyuridine- 

 5 '-triphosphate was used in place of the corresponding thymidine com- 

 pound, it was demonstrated that deoxyuridine was bound in a 3 '-5' phos- 

 phodiester linkage with each of the deoxyribonucleosides of adenine, 

 guanine, cytosine, and uracil. 



91 M. J. Bessman, I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms, and A. 

 Romberg, Proc. Natl. Acad. Sci. U. S. 44, 633 (1958). 



