I. REPLICATION OF DNA IN CELL-FREE SYSTEMS 57 



Josse and Kornberg, 1962). The only activity not yet found is the enzyme 

 which forms diglycosyl-HAIC units in T2 DNA. 



UDP-glucose (Caputto et al., 1950) has been shown to be the 

 coenzyme reciuii'ed for tlie transfer of glucose to the HAIC residues in 

 each of the reactions tested (Kornberg et al., 1959). The enzymes will 

 not glucosylate the free deoxyribonucleotide or deoxyribonucleositle 



TABLE XXVIII 

 Analysis of HMC"-Residues in Bacteriophage DNA* 



" HMC = h3'drox3'meth3'lc3'tosine. 

 * Data from Lehman and Pratt (1960). 



triphosphate, but require HMC residues in intact DNA. Conversely, 

 synthetic glucosyl hydroxymethyldeoxycytidine triphosphate in the pres- 

 ence of the other three triphosphates is much less effective than the 

 non-glucosylated triphosphate in supporting DNA synthesis in extracts 

 of infected cells (Koerner et al., 1960). The generalized reaction for the 

 a-glucosyl and /?-monoglycosyl transferase may be formulated: 



UDP-glucose + HMC-DNA -^ UDP + glucosyl-HMC-DNA 



The HMC-DNA used as acceptor for the glucosyl units is prepared by 

 incubating E. coli polymerase with 5-hydroxymethyldeoxycytidine tri- 

 phosphate, the other three deoxyribonucleoside triphosphates, Mg ions, 

 and primer DNA. Several different types of primer, including DNA from 

 E. coli, bacteriophage, and calf thymus have been used with equal 

 success. Alternatively, naturally occurring HMC DNA may be used as 

 acceptor provided the heterologous transferase is being studied. For ex- 

 ample, T2 DNA (containing 25% unglucosylated HMC residues) is a 

 good substrate for T4 or T6 a-transferase but will not accept glucose 

 using the purified T2 transferase. As a general rule, no transferase will 

 catalyze the further glucosylation of its homologous DNA. 



The T2, T4, and T6 a-glucosyl transferases are three very closely 

 related proteins which have many properties in common. They are differ- 

 ent in certain respects as is evident from an inspection of Table 

 XXIX. The T4 /^-transferase is readily distinguishable from any of the 

 a-transferases. 



