ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. VI . INFLUENCE OF BACTERIOPHAGE Ti? ON THE SYNTHETIC PATHWAY IN HOST CELLS* BY ARTHUR KORNBERG,

Information now available about DNA‘ synthesis by Escherichia coli e n z y r n e ~ ~ ~ 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.6 This general problem poses several rather specific questions which may be summarized as follows : 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 hydroxymethylates 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? 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 triphosphate from the site of polymerase action? The DNA’s of T2, T4, and T6 contain glucose linked to the hydroxymethyl groups of the HMC in characteristic ratios;# 9* lo although it is clear that in T2 and T6 some of the HMC groups contain no gluco~e.~ 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 nonglucosylated HMC nucleotides. Is there an alternative mechanism involving direct glucosylation of the DNA even though direct substitutions on intact DNA have been hitherto unknown? Following phage T2 infection there is a temporary halt followed by a resumption 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 DNAsynthesizing activity.I2 What are the altered conditions for assay of DNA synthesis in infected cell extracts which would elicit the high levels of activity expected from the physiologic studies? We have explored these questions and have found that following infection with phage T2 several new enzymes appear.13 These are (1) an enzyme which phosphorylates dHMC-5-P, leading to the synthesis of hydroxymethyldeoxycytidine triphosphate (dHMC-TP), (2) an enzyme which removes the terminal pyrophosphate 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.