20 MAUUICK J. UESSMAX 



vestigation (cf. Shii^ar, I9()()i hut it is clear tliat there are two distinct 

 contributions to the over-all hyjujchroniicity of DNA solutions. The 

 first is maintained through the hydrogen bonding between the base pairs 

 in the twin-stranded Watson-Crick model and accounts for approxi- 

 mately 50% of the total hypochromicity. The remainder is maintained 

 through the jihosphodiester bridges between the individual nucleotides 

 (Sinsheimer, 1954; de Garilhe and Laskowski, 1956; Michelson, 1958). 

 An illustration of this is shown in Fig. 5. 



The decrease in extinction during polymerization of mononucleotides 

 can be made the basis of an assay for DNA synthesis. The method is not 

 suitable for measurements of small increments of DNA since the effect 

 is relatively small and in addition to the absorption of the nucleotides 

 themselves there is the contribution of the primer DNA. The method has 

 been used successfully in measuring unprimed reactions and reactions in 

 which extensive synthesis of polymer occurs (see Section IV,G). 



B. REQUIREMENTS OF THE REACTION 



1. Forward Reaction 



The synthesis of DNA catalyzed by purified fractions of E. coli 

 polymerase requires the presence of all four of the deoxyribonucleoside 

 triphosphates oonimonly found in DNA, Mg ions, and a highly polymer- 

 ized preparation of DNA (Bessman et at., 1958a). In Table VI, the 



TABLE VI 

 Requirements for Deoxynucleotide Incorporation' into DNA"* 



System mMinoles 



Complete system . 50 



Omit dCTP, dGTP, dATP <0.01 



OmitdCTP <0.0I 



Omit dGTP <0.01 



Omit dATP <0.01 



Omit Mg++ <0.01 



Omit DNA <0.01 



DNA pretrcated with DNase <U . 1 



" P>om Bessman et al. (1958a). 



*" The complete system contained 5 m^moles of dTTP (dTP'^PP, 1.5 X 10* cpm per 

 A/mole), dATP, dCTP, and dGTP, 1 Mmole of MgCU, 20 fimoles of glycine buffer, pH 9.2, 

 10 fig of DNA, and 8 /ig of "polymerase" fraction V in a final volume of 0.30 ml. The 

 incul)ation was carried out at 37°C for 30 minutes. 



effect of omitting any one or combinations of these constituents is 

 reported. Within the limits of this particular assay, omission of any one 



