138 H. GOBIND KHORANA 



By using thymidine-5'-triphosphate labeled in the ring (C 14 ) and in the 

 pyrophosphate (P 32 ), good stoichiometry was demonstrated between the 

 nucleotide incorporated and inorganic pyrophosphate released. 86 



b. The Chemical and Physical Properties of the Synthetic Products 



Net synthesis of polymer has provided material for physicochemical 

 studies and comparison with the DNA used as primer. The enzymically 

 synthesized product has essentially the same physical properties as DNA 

 prepared from calf thymus. 88 Thus, the sedimentation and viscosimetric 

 behavior are quite similar. The observed greater polydispersity of the en- 

 zymic product was ascribed to the action of contaminating nucleases in 

 the polymerase preparation. From the data, several synthetic products 

 were concluded to have average molecular weights in the neighborhood of 

 5 to 6 million and to have highly ordered rigid structures with effective vol- 

 umes greater than would be expected from single polynucleotide chains. 

 The behavior on heating at 100°, namely the collapse of macromolecular 

 structure, was again similar to that of calf thymus DNA. Furthermore, 

 degradation with pancreatic deoxyribonuclease gave the typical hyper- 

 chromic effect shown by DNA 89 (Volume I, Chapter 14). 



The evidence for the nature of internucleotide linkages in the enzymically 

 synthesized products comes from the degradation with pancreatic deoxy- 

 ribonuclease. 86 The extent and pattern of degradation is very similar to 

 that previously established for DNA. Furthermore, when one of the four 

 nucleotide substrates, namely thymidine-5'-triphosphate, labeled with C 14 

 was used, degradation by pancreatic deoxyribonuclease gave in the appro- 

 priate region of the ion exchange elution diagrams labeled dinucleotides 

 which were identified as deoxycytidylyl-(5' — > 3')-thymidylic-(5') acid and 

 thymidylyl-(5' — * 3')-thymidylic-(5') acid. The labeled starting mate- 

 rial was thus incorporated in typical C 3 '-C 5 ' internucleotide linkages in 

 polynucleotide chains. From this and further experiments, as well as the 

 evidence from the limited synthetic reaction discussed below, it may be 

 concluded that the synthetic reaction results in the formation of the inter- 

 nucleotide linkages known to exist in DNA. 



c. Reversal of the Synthetic Reaction* 6 



Inorganic pyrophosphate in the reaction mixture when present in the 

 same concentration as the deoxyribonucleoside triphosphates (1.6 X 10 5 

 M) has no effect on the rate of the synthetic reaction. However, at approxi- 

 mately one hundred times the above concentration the synthetic rate is 



88 H. K. Schachman, I. R. Lehman, M. J. Bessman, J. Adler, E. S. Simms, and A. 

 Kornberg, Federation Proc. 17, 304 (1958). 



89 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 



