I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 9 



originally reported an increase in this activity in extracts of cells infected 

 with bacteriophage T6 and suggested the name "thymidylate synthetase" 

 since it was not known whether one or more enzymes was required to 

 transfer the 1-carbon unit and cany out the reductive step (Flaks and 

 Cohen, 1959b). These same investigators (1959b) demonstrated a 7-fold 

 increase in thymidylate synthetase after infection with bacteriophage 

 T2 and showed that the conversion of deoxyuridylate to thymidylate 

 required formaldehyde and tetrahydrofolic acid; it did not proceed 

 through 5-hydroxymethyldeoxyuridylate as an intermediate. Earlier ex- 

 periments of Earner and Cohen (1954), in which they showed that a 

 thymine-requiring strain of E. coli, IST", could synthesize thymine after 

 infection with bacteriophage, can be explained by later experiments of 

 these same authors (1959) in which they demonstrated that thymidylate 

 synthetase (missing in 15T-) is induced by bacteriophage infection. This 

 would support the idea that the infecting phage particle carries in the 

 information for the induction of thymidylate synthetase, and Cohen 

 (1961) investigated whether the enzyme foraied after infection is the 

 same as the enzyme in the normal cell. A comparison of thymidylate 

 synthetase in extracts of E. coli B, Bt" infected with T2 and Bt- infected 

 with T5 showed that the enzymes from all three sources were inhibited 

 to the same extent by varying concentrations of 5-flu6rodeoxyuridylate 

 or D-arabinofuranosyl-5-fluorouracil-5'-phosphate, two powerful inhibi- 

 tors of thymidylate synthetase. The active sites of the enzymes from 

 these three sources are thus indistinguishable by this test. Recently, 

 however, Greenberg et al. (1962b) have demonstrated that thymidylate 

 synthetase from T2-infected E. coli has different chromatographic 

 properties from the thymidylate synthetase of uninfected cells. 



Cohen et al. (1958) have shown that 5-fluorodeoxyuridylate is a 

 powerful non-competitive inhibitor of thymidylate synthetase, and at a 

 concentration of 2 X 10"*^ M (a ratio of substrate to inhibitor of 1000) 

 from 35-55% inhibition was observed. Heidelberger et al. (1957) have 

 reported the anti-tumor activity of 5-fluorouracil and presented evidence 

 for the inhibition of thymine synthesis in whole cells by this analog. 

 The inhibition of thymidylate synthetase by 5-fluorodeoxyuridylate 

 could explain this observation. 



As mentioned previously (Section I,A), Bertani et al. (1961) have 

 indicated that the conversion of uridylate to deoxyuridylate occurs at 

 the diphosphate level. The bulk of the evidence indicates that the 

 methylation of deoxyuridylate to thymidylate occurs at the monophos- 

 phate level. This would mean that deoxyuridine diphosphate would have 

 to be converted to deoxyuridine monophosphate before methylation. 

 Bertani et al. (1961) have found that deoxy-UDP is phosphoiylated to 



