I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 15 



Maley and Ochoa (1958), who described the properties of the deoxy- 

 cytidylate kinase in Azotobacter vinelandii. This enzyme was purified 

 800-fold over the crude extract and was shown to catalyze the following 

 reaction: 



dCMP + ATP -' (K^DP + A DP 



The other three nucleotides (dGMP, dTMP, or dAMP) are not sub- 

 strates for this enzyme. 



The stoichiometry of the reaction is shown in Table \. In Expt. 1, a 

 less purified enzyme was used and it can be seen that botli dCDP and 

 dCTP were formed probably due to the presence of a nucleoside diphos- 

 phate kinase which is known to be present in Azotobacter. In the more 

 purified fractions (Expts. 2 and 3) only the diphosphate was formed. The 

 same enzyme appears to catalyze the phosphorylation of the ribo 

 compound, cytidylic acid, since the ratio of the activities toward the 

 two substrates remains constant during the purification. Cytidylate is 

 actually phosphoiylated at about 1.5 times the rate of deoxycj'tidylate 

 by this enzyme at equal concentrations of the two compounds. 



" From Maley and Ochoa (1958). 



*" All samples contained 100 Mmoles of tris buffer, pH 7.5, and 20 yumoles of MgCh 

 in a final volume of 1 .0 ml and were incubated at 30°C. The remaining components and 

 conditions were as follows: Expt. 1: 9 Mmoles of ATP, 9 /^moles of dCMP, and 10 ng of 

 enzyme of specific activity 60 with incubation for 15 hours; Expt. 2: 10 /xmoles of ATP, 

 5.8 /xmoles of dCMP, and 2.3 Mg of enzj'me of specific activity 175 with incubation for 

 3 hours; Expt. 3: 4.5 ^moles of ADP, 1.1 /imoles of dCDP, and 2.3 ^g of enzyme of specific 

 activity 175 with incubation for 18 hours. Results are expressed as /imoles decrease or 

 increase in reactants and products. 



We have purified a deoxyguanylate kinase (M. Bessman, unpub- 

 lished) from extracts of E. coli approximately 400-fold which appears 

 to catalyze the following reaction. 



dGMP + ATP ^ dGDP + ADP 



Ion exchange chromatography reveals that more than 95% of the 

 phosphoiylated product is the diphosphate. Again, ribo-GlVIP is also 

 phosphoiylated but dCMP and dT]\IP are not. 



Both these purified enzymes (deoxycytidylate and deoxyguanylate 

 kinases) phosphoiylate the ribo- as well as deoxyribonucleotide. The 



