Vol. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 783 



interesting question regarding T2 infection of E. coli is how the T2 DNA seems 

 to preempt from the host DNA the primer function for the polymerase system. 

 The present studies can explain why DNA synthesized by the phage T2-E. coli 

 system contains hydroxymethylcytosine rather than cytosine, but not why the 

 base composition is that of phage rather than that of host DNA. An observation 

 reported in this paper which may bear on this question is the dependence of the 

 augmented polymerase activity of the infected cell extracts on a physically altered 

 DNA primer. When "native" calf thymus or phage T2 DNA was used, the 

 polymerase values of infected and normal cell extracts were similar. However, 

 with DNA samples heated at pH 9, the values for dHMC-TP incorporation with 

 infected cell extracts were increased 12-fold, while the normal cell values were 

 unaffected or even depressed. It is not clear as yet whether infection has resulted 

 in formation of a new type of polymerase or in an increase of the normal poly- 

 merase and new factors which modify it. Further studies to clarify this question 

 may lead to a better insight into how the injected phage DNA proves to be the 

 chosen primer for this system. 



SUMMARY 



1. Extracts of E. coli infected with bacteriophage T2 have been shown to 

 contain three enzymes which are undetectable in extracts of uninfected or in T5- 

 infected cells. These are : (a) an enzyme which phosphorylates hydroxymethylde- 

 oxycytidine 5-phosphate, leading to the synthesis of the triphosphate, (6) an enzyme 

 which removes the terminal pyrophosphate group specifically from deoxycytidine 

 triphosphate, and (c) an enzyme which transfers glucose from uridine diphosphate 

 glucose directly to the hydroxymethylcytosine of certain DNA's. 



2. These new enzymes can account for (a) the availability of the triphosphate 

 of hydroxymethyldeoxyc3nDidine for the enzymatic synthesis of T2 DNA, (6) 

 the absence of deoxycytidylate from T2 DNA, and (c) the presence of glucose on a 

 fixed fraction of the hydroxymethylcytosine residues in DNA. 



3. The DNA-polymerizing enzyme assayed with hydroxy methyldeoxycytidine 

 triphosphate in place of deoxycytidine triphosphate reveals about a 12-fold increase 

 in activity after T2 infection. Increases, after T2 infection, in the levels of thymine 

 and guanine deoxynucleotide phosphorylating enzymes (by 20-45 fold) bring 

 their activities up to the level of the adenine deoxynucleotide phosphorylating 

 enzyme which is unchanged ; the level of the cytosine deoxynucleotide phosphorylat- 

 ing enzyme remains at a low level. 



4. After T5 infection, levels of the thymine, guanine, and cytosine deoxynucleo- 

 tide phosphorylating enzymes increase by 10-40 fold, bringing their activities up 

 to the level of the adenine deoxynucleotide phosphorylating enzyme, which in- 

 creases about 2-fold. 



5. The new enzymes and the increases in level of the enzymes occurring in 

 normal cells are first detectable about 4 min after infection with phage T2 and 

 about 10 min after infection with phage T5. These results are consistent with 

 previously published studies which have indicated with the use of inhibitors of 

 protein synthesis that viral DNA synthesis requires a preliminary period of pro- 

 tein synthesis. 



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