I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 7 



serine can be explained by the observation that the active 1 -carbon 

 unit wliich ultiniately becomes the methyl group of thymine can 

 be derived from the /3-carbon of serine (Ehvyn and Sprinson, 1954). 

 The requirement for serine could be replaced by synthetic hydroxy- 

 methyltetrahydrofolic acid which suggested that this was the active 

 1 -carbon intermediate generated in this system. These observations 

 were extended to animal tissues by Phear and Greenbcrg (1957) and 

 Humphreys and Greenberg (1958) and to a serineless mutant of 

 E. coll by Birnie and Crosbie (1958). The bulk of the evidence impli- 

 cates deoxyuridine monophosphate as the immediate precursor of thy- 

 midylate in these systems. Blakely (1957), however, has suggested from 

 his experiments that deoxyuridine rather than the corresponding nucleo- 

 tide is the acceptor of the methyl group. 



TABLE IV 



Stoichiometry of Thymidylate Formation from Deoxyuridylate"'^ 



" From Wahba and Friedkin (1961). 



'' The incubation mi.xture consisted of 0.040 ^mole of dUMP (labeled with P'^, 

 80,000 cpm); 0.15 /xmole of dl, L-tetrahydrofolate; and enzj^me (0.2 mg of protein) in 

 1 ml of a mi.xture containing tris-HCl buffer, pH 7.4, 40 pinoles; 2-mercaptoethanol, 100 

 )umoles; ethylenediaminetetraacetate, 1.0 pinole; MgCU, 25 pmoles; and formaldehyde, 

 15 ^moles. 



In addition to its role as carrier of the 1-carbon unit, tetrahydrofolic 

 acid has been implicated as the reducing agent in the methylation reac- 

 tion (Friedkin, 1959a; Humphreys and Greenberg, 1958). Direct evidence 

 for this has been presented by Friedkin (1959a), who has reported the 

 transfer of tritium from tritiated tetrahydrofolic to thymidylic acid. In 

 addition, Wahba and Friedkin (1961) have recently described the 

 stoichiometric oxidation of tetrahydrofolic to dihydrofolic acid during 

 the synthesis of thymidylate from dcoxyuridylate. For each mole of 

 thymidylate formed, one mole of dihydrofolate appeared. These results 

 are reported in Table IV. Friedkin (1959b) has proposed a scheme for 

 the methylation of deoxvuridvlate bv N^'-N'" methvlenetetrahvdrofolic 



