33. NUCLEIC ACIDS OF THE BACTERIAL VIRUSES 213 



formaldehyde plus tetrahydro folic acid converts dCMP to dHMP (deoxy- 

 cy tidy late hydroxymethylase). Cohen had previously demonstrated that 

 the hydroxymethyl group could be derived from the (8-carbon of serine, 102 

 but not from methionine. 11911 This deoxycytidylate hydroxymethylase could 

 not be demonstrated in extracts of normal cells or of bacteriophage, nor 

 could it be inhibited by mixtures of extracts of normal and infected cells. 

 If the phage infection was made in the presence of the inhibitor 5-methyl- 

 tryptophan, no enzyme was made until the inhibition was released by the 

 addition of tryptophan. 



Romberg et al., 77 and Somerville and Greenberg 120 have demonstrated 

 the appearance of a 5-hydroxymethyldeoxycytidylic acid kinase which con- 

 verts the monophosphate (dHMP) to the triphosphate (dHTP), which is 

 the apparent DN A precursor. Studies of the levels of the other deoxyribo- 

 nucleotide kinases during infection indicate that the levels of deoxyguanylic 

 kinase and thymidylic kinase rise markedly (twenty- to fortyfold), the level 

 of deoxyadenylic acid kinase which is peculiarly high in uninfected cells 

 remains high, and the level of deoxycy tidy lie acid kinase remains low. 



A particularly interesting enzyme which appears during T2 infection is a 

 deoxycy tidy lie triphosphatase, 77, 121 which converts deoxycytidylic triphos- 

 phate (dCTP) to the monophosphate by hydrolysis of the pyrophosphate 

 link. The presence of this enzyme prevents the accumulation of any dCTP 

 and thereby explains the complete substitution of cytosine by 5-hydroxy- 

 methylcytosine in phage DNA. The DNA polymerase 122 which increases 

 some twelvefold in activity during infection, does not itself discriminate 

 between dCTP and dHTP. 



Another interesting enzyme, described by Romberg et al. in T2 infected 

 cells, glucosylates a fraction of the hydroxymethyl groups of 5-hydroxy- 

 methylcytosine after the incorporation of this nucleotide into macromolecu- 

 lar DNA. The glucose is transferred from uridine diphosphate glucose 

 (UDPG). 



In T6 phage, most of the hydroxymethyl groups are coupled to a diglu- 

 cose and an additional enzyme has been isolated from T6 infected cells 

 which adds a second glucose, again from UDPG (Romberg, personal com- 

 munication). 



These new enzymes, or the increases in existing enzymes, begin to appear 

 about 3 to 4 minutes after infection with the phage. 



118 J. G. Flaks and S. S. Cohen, J. Biol. Chem. 234, 1501 (1959). 



119 J. G. Flaks, J. Lichtenstein, and S. S. Cohen, J. Biol. Chem. 234, 1507 (1959). 

 119a M. Green and S. S. Cohen, J. Biol. Chem. 225, 387 (1957). 



120 R. Somerville and G. R. Greenberg, Federation Proc. 18, 327 (1959). 



121 J. F. Koerner and M. S. Smith, Federation Proc. 18, 264 (1959). 



122 I. R. Lehman, M. J. Bessman, E. S. Simms, and A. Romberg, ./. Biol. Chem. 233, 

 163 (1958). 



