31. SYNTHESIS OF POLYNUCLEOTIDES 



137 



1. Microorganisms 



The studies of Romberg and co-workers with a system prepared from 

 Escherichia coli (Strain B or ML 30) have provided the lead and all the 

 definitive information that is available on the bacterial systems. The sys- 

 tem has already made it possible to test many of the tenets of the well- 

 known Watson-Crick 84 structure for the deoxyribonucleic acids (DNA) and 

 it appears that in the near future much more insight into the chemistry of 

 the biosynthesis of DNA and its replication will be gained from these stud- 

 ies. 



a. Basic Requirements of the Enzymic System? 5 - 86 



The enzyme which has been called polymerase, has been purified 2000- 

 4000-fold. 85 It is inactivated upon heating at neutral pH's and is inhibited 

 by high concentrations of salt. Inhibition of DNA synthesis by sodium ions 

 has also been reported by Walwick and Main. 87 The requirements for 

 maximal activity are the presence of the 5'-triphosphates of all the four 

 deoxyribonucleosides (thymidine, deoxycytidine, deoxyadenosine, and 

 deoxyguanosine), magnesium ions, and a primer. DNA from a variety of 

 sources can serve as the primer. Under these conditions, the reaction may 

 be assayed by conversion of the acid-soluble mononucleotides to an acid- 

 insoluble polymer and extensive net synthesis (2-20 times the amount of 

 the primer) can be realized. 86 Omission of any one of the triphosphates, or 

 of either of the other components, or digestion of primer DNA with crystal- 

 line pancreatic deoxyribonuclease (however, see below, Sections g and i) 

 reduces the reaction to below 1 %. 86 The triphosphates cannot be replaced 

 by the corresponding diphosphates. The over-all reaction may therefore be 

 represented as follows: 



"dAP" 



dATP 



+ 



dGTP 



+ 



dCTP 



+ 



TTP 



+ DNA ^ DNA- 



dGP 



dCP 



TP 



+ in PP 



84 J. D. Watson and F. H. C. Crick, Nature 171, 737, 964 (1953). 



85 I. R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg, J. Biol. Chem. 233, 

 163 (1958). 



86 M. J. Bessman, I. R. Lehman, E. S. Simms, and A. Romberg, J. Biol. Chem. 233, 

 171 (1958). 



87 E. R. Walwick and R. K. Main, U.S. Navy Radiological Defense Laboratory, 

 Tech. Rept. No. 319 (1959). 



