I. REPLICATION OF DNA IN CELL-FREE SYSTEMS d 



and a requirement for primer DNA will l)e considered in this chapter. 

 The influence of primer on the synthetic reaction is a necessary but 

 certainly not a sufficient condition for replication since, as will be 

 discussed later, there are some situations in which the synthetic DNA 

 reflects the composition and structure of the added primer, and other 

 primer-dependent reactions in which the product is clearly different 

 from the primer. 



II. Synthesis of Deoxyribonucleotides 



A. ORIGIN OF THE DEOXYRIBOSIDES 



Recent experiments with cell-free systems support the earlier indica- 

 tions from tracer experiments in the whole animal that deoxyribosyl 

 compounds are synthesized by a direct reduction of some ribosyl deriva- 

 tives. Hammarsten et al. (1950) suggested that the N^^-labeled deoxy- 

 cytidine they isolated from rat liver DNA arose by a direct conversion 

 of the injected N^^-cytidine without cleavage of the ribosidic linkage, 

 since it was known from the results of several laboratories that free 

 cytosine is not a good precursor of DNA cytosine. Experimental evidence 

 in support of this contention was furnished by the work of Rose and 

 Schweigert (1953), who injected C"-cytidine, labeled in both the base 

 and the ribose moieties, into rats and found that the ratio of counts in 

 the base and deoxyribose in the DNA deoxycytidine was the same as 

 the ratio of counts in the base and ribose of the injected nucleoside. 

 Either cytidine was converted to deoxycj^tidine without cleavage of the 

 ribosidic linkage, or cleavage did occur and the pool sizes of the free 

 cytosine and ribose in the rat were exactly the same. The latter possi- 

 bility seems too remote to have been seriously entertained. These results 

 were confirmed by Roll et al. (1956) and Reichard (1957) and extended 

 to include chick embiyo (Reichard, 1958, 1959), Ehrlich ascites cells 

 (Edmunds, 1958) and Neurospo7'a (McNutt, 1958a). Cell-free extracts 

 of chick embryo (Reichard, 1959, 1960), Escherichia coli (Reichard and 

 Rutberg, 1960; Reichard et al, 1961), and Novikoff hepatoma (Moore 

 and Hurlbert, 1960) were shown to carry out the reduction of ribosyl 

 compounds to deoxyribosyl derivatives and, as is usually the case, it has 

 been through the use of purified, cell-free systems that insight into the 

 mechanism of the interconversion has become possible. Reichard et al. 

 (1961) had suggested from isotope dilution experiments and kinetic data 

 that the conversion of cytidine to deoxycytidine nucleotides occurred at 

 the diphosi:)hate level and required ATP, ^Vlg'^^, and TPN. Bertani et al. 

 (1961), using a fraction purified 100-200- fold from crude extracts of 

 Escherichia coli. have presented clear evidence that the reduction occurs 



