ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. VI. 



INFLUENCE OF BACTERIOPHAGE T2 ON THE SYNTHETIC 



PATHWAY IN HOST CELLS* 



By Arthur Kornberg, Steven B. Zimmerman,^ S. R. Kornberg, and 



John Josse* 



DEPARTMENT OF MICROBIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS 



Communicated April 20, 1959 



Information now available about DNA^ synthesis by Escherichia coli enzymes^"* 

 has encouraged an inquiry into the biochemical basis for the observation that a 

 phage-infected E. coli cell ceases to produce its own DNA and makes instead the 

 DNA characteristic of the infecting phage. ^ This general problem poses several 

 rather specific questions which may be summarized as follows : 



1. T2, T4, and T6 DNA differ from the DNA of E. coli, as well as from that 

 of other sources which have been examined, in containing hydroxymethylcytosine 

 (HMC) but no cytosine.* Flaks and Cohen^ have already shown that within 

 several minutes after infection by phage T2, T4, or T6, a new enzyme which hy- 

 droxy methylates deoxycytidine 5 '-phosphate is produced. Is there an enzyme 

 for converting the resulting dHMC-5-P to the triphosphate level in order to provide 

 a functional substrate for DNA synthesis? 



2. With respect to the exclusion of cytosine from the DNA of phage T2, T4, and 

 T6, is there a mechanism in the infected cell for removal of deoxycytidine triphos- 

 phate from the site of polymerase action? 



3. The DNA's of T2, T4, and T6 contain glucose linked to the hydroxymethyl 

 groups of the HMC in characteristic ratios,*' ^' '" although it is clear that in T2 

 and T6 some of the HMC groups contain no glucose.^ According to our present 

 understanding of DNA synthesis,^ it is difficult to conceive how these constant 

 ratios are achieved if the incorporation were to occur via glucosylated and non- 

 glucosylated HMC nucleotides. Is there an alternative mechanism involving 

 direct glucosylation of the DNA even though direct substitutions on intact DNA 

 have been hitherto unknown? 



4. Following phage T2 infection there is a temporary halt followed by a resump- 

 tion of DNA synthesis at about 5 times the rate shown by the uninfected cell. " 

 However, measurements with extracts of infected cells, using standard substrates, 

 revealed much diminished rather than the anticipated augmented levels of DNA- 

 synthesizing activity.^- What are the altered conditions for assay of DNA syn- 

 thesis in infected cell extracts which would elicit the high levels of activity ex- 

 pected from the physiologic studies? 



We have explored these questions and have found that following infection with 

 phage T2 several new enzymes appear. ^^ These are (1) an enzyme which phos- 

 phorylates dHMC-5-P, leading to the synthesis of hydroxymethyldeoxycytidine 

 triphosphate (dHMC-TP), (2) an enzyme which removes the terminal pyro- 

 phosphate group from dCTP, and (3) an enzyme which transfers glucose from 

 UDPG directly to the HMC in DNA. Measurements of DNA synthesis, using 

 dHMC-TP in place of dCTP, revealed about a 12-fold increase in activity in 

 extracts of infected cells over the levels observed in uninfected cell extracts. 



Reprinted by permission of the authors and the National Academy 



of Sciences from the Proceedings of the National Academy of 



Sciences, 45 (6), 772-785, June, 1959. 



125 



