36. BIOSYNTHESIS OF PYRIMIDINB NUCLEOTIDES 345 



Cohen et al. lbb have investigated the role of 5-hydroxymethyluracil, 5-hy- 

 droxymethylcytosine, and the corresponding deoxyribonucleosides in 

 bacterial and bacteriophage DNA-pyrimidine synthesis using the isotopic 

 competition technique. No evidence for the utilization of any 5-hydroxy- 

 methylpyrimidine derivative was obtained. 4,5-Dihydro-5-hydroxymethyl- 

 uracil, 4,5-dihydro-5-hydroxymethylcytosine, and the corresponding de- 

 oxynucleosides do not support 156 the growth of the thymine-less mutant, E. 

 coli 15T-. It would appear that any involvement of 5-hydroxymethyl- and 

 4,5-dihydro-5-hydroxymethylpyrimidine derivatives in thymine ring bio- 

 synthesis must occur at the level of the nucleotide. 



A possible alternative mechanism not outlined in Fig. 5 involves a reduc- 

 tion of dUMP to the 4,5-dihydro derivative prior to condensation with 

 JV 5 ,Af 10 -methylene-FH4 . There is, however, considerable evidence contra- 

 indicating the role of dihydropyrimidine derivatives in TMP synthesis. 

 Thus dihydrodeoxyuridine (or dihydrodeoxycytidine) is not utilized 155 for 

 DNA-thymine synthesis in E. coli B, E. coli 15T- (a thymine-less mutant), 

 or for viral thymine and 5-hydroxymethylcytosine in T6r + grown in E. coli 

 B or E. coli Bu- (a uracil-less mutant). Evidence that dihydrodeoxyuridine- 

 2-C 14 penetrates the coli cell is provided by the isolation of a phosphorylated 

 derivative (probably the corresponding 5'-nucleotide) from the acid-soluble 

 fraction of E. coli 15T-. Blakley 150 has similarly reported the noninvolve- 

 ment of dihydrodeoxyuridine (and dihydrouridine) in thymidine synthesis 

 in his cell-free thymus system. This evidence is not, however, unequivocal 

 as the "methylation reaction" may well be occurring at the level of the nu- 

 cleotide in a system possessing no kinase activity towards the dihydro- 

 deoxy nucleoside . 



E. coli thymidylate synthetase (unlike 5-hydroxymethyldeoxycytidylate 

 synthetase) is inhibited powerfully and irreversibly by 5-fluorouracil deoxy- 

 ribonucleoside-o'-phosphate. 157 



Integration of the pathway of de novo synthesis of UMP with the thy- 

 midylate synthetase reaction has been achieved recently by Reichard 100 

 w T ho has described briefly the formation of dUMP from UMP by a soluble 

 enzyme of chick embryo homogenates. No evidence for a pentose intercon- 

 version at the nucleoside level was obtained (cf. Grossman 158 who has re- 

 cently reported without details that a dithiol-activated soluble enzyme of 

 Salmonella tymphimurium LT-2 effects a conversion of uridine to deoxyuri- 



155 S. S. Cohen, J. Lichtenstein, H. D. Barner, and M. Green, J. Biol. Chem. 228, 611 



(1957). 

 186 M. Green, H. D. Barner, and S. S. Cohen, /. Biol. Chem. 228, 621 (1957). 



157 S. S. Cohen, J. G. Flaks, H. D. Barner, M. R. Loeb, and J. Lichtenstein, Proc. 

 Natl. Acad. Sci. U.S. 44, 1004 (1958). 



158 L. Grossman, Federation Proc. 17, 235 (1958). 



