I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 27 



throughout the molecule by hydrolysis of phosphodiester linkages, thus 

 facilitating strand separation and enhancing priming efficiency. 



With some highly purified fractions of E. coli polymerase, native 

 DNA acts veiy poorly as a primer compared to heated DNA or DNA 

 slightly hydrolyzed with DNase. This raised the question (Kornberg, 

 1960) of whether native DNA (double-stranded) can function at all as 

 primer. To test this possibility rigorously, E. coli polymerase prepara- 

 tions free of nuclease activity must be used; unfortunately, at present, 

 none are available. 



E. NATURE OF THE PRODUCT 



1. Chemical Structure 



The deoxynucleotides incorporated into DNA by E. coli polymerase 

 are present in 3'-5' phosphodiester linkage (Bessman et al., 1958a). A 

 product labeled with C"-thymidylate was digested with deoxyribonucle- 

 ase and chromatographed according to Sinsheimer (1954). Two dinucleo- 

 tides, cytidylate-thymidylate and thymidyl-thymidylate, were isolated 

 and characterized as dinucleotides with typical 3'-5'-phosphodiester 

 bridges. Thus it is clear that covalent linkages are fomied during the 

 incorporation and the four deoxyribonucleotides add to each other. This 

 rules out the possibility, for example, that homopolymers of the indi- 

 vidual deoxyribonucleotides are the sole products. Besides having the 

 nonnal internucleotide linkages, the product reacts with diphenylamine 

 to give the characteristic color for deoxyribose (Dische, 1955). This is 

 shown in Table XI, where the values for deoxy ribonucleotide incorpora- 



" From Bessman et al. (1958a). 



tion (as measured by P^-), ultraviolet extinction, and deoxyribose con- 

 tent of a synthetic DNA are compared. 



