I. REPLICATION OF DNA IN CELL-P^REE SYSTEMS 17 



two cases cited above which were studied with purified enzymes (dCMP 

 and dGMP kinases from E. coli) the diphosphate accumulates in the 

 reaction mixture and no triphosphate is fomied. However, as discussed 

 above, crude extracts of E. coli, regenerating rat liver, leukemic cells, 

 Ehrlich ascites cells, thymus tissue, etc., form predominantly triphos- 

 phates in the presence of ATP and a deoxyribonucleoside monophosphate. 

 These two observations taken together mean there must be an enzyme (s) 

 which catalyzes the phosphorylation of the deoxyribonucleoside diphos- 

 phates formed as intermediates during the incubation. However, no 

 specific deoxyribonucleoside diphosphate kinases have been reported, 

 with the possible exception of the dTDP kinase alluded to in the report 

 of Weissman et al. (1960). It is not unlikely that the nucleoside diphos- 

 phate kinases described by Berg and Joklik (1953) and Krebs and Hems 

 (1953), which catalyze the phosphorylation of the ribonucleoside diphos- 

 phates, also react wdth the corresponding deoxy compounds: 



(deoxy)micleoside diphosphate + ATP ;=i (deoxy) nucleoside triphosphate + ADP 



Kirkland and Turner (1959) have shown that, in addition to the phos- 

 phorylation of IDP and UDP originally demonstrated by the above 

 investigators, purified extracts of peas also phosphoiylate CDP and 

 GDP. 



Davidson (1959), in a reinvestigation of the specificity of pyruvate 

 kinase, has demonstrated that GDP can replace ADP in the reaction. 

 It is possible that dGDP also could be phosphorylated in this system 

 since Klenow and Anderson (1957) presented evidence for the replace- 

 ment of ADP by dADP using crystalline pyruvate kinase from rabbit 

 muscle. It is clear that the specificity of these enzymes with respect to 

 the deoxy compounds should be investigated. 



IV. Synthesis of DNA by Escherichia coli Polymerase 



A. MEASUREMENT OF DNA SYNTHESIS 



1. Acid Insolubility 



The insolubility of DNA in dilute acids affords a convenient means 

 of separating the macromolecule from low molecular weight deoxy- 

 nucleotide derivatives which are soluble in acid solution. For the ensuing 

 discussion, we shall define "DNA synthesis" to mean the incorporation 

 of acid-soluble deoxyribonucleotide derivatives into covalent linkage in 

 an acid-insoluble product. This is purposely a broad definition which 

 includes "exchange reactions" if they occur as well as reactions in which 



