784 BIOCHEMISTRY: KORNBERG ET AL. Proc. N. A. S. 



* This investigation was supported by research grants from the National Institutes of Health 

 of the Public Health Service and the National Science Foundation. 



t National Science Foundation Predoctoral Fellow. 



X Fellow of the National Foundation. 



' The abbreviations used in this report are: cpm, counts per minute; ATP, adenosine tri- 

 phosphate; dA-5-P, deoxyadenosine 5 '-phosphate; dC-5-P, deoxycytidine 5 '-phosphate; dG-5-P, 

 deoxyguanosine 5'-phosphate; dHMC-5-P, hydroxymethyldeoxycytidine 5'-phosphate; dT-5-P, 

 deoxy thymidine 5 '-phosphate; dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine tri- 

 phosphate; dGTP, deoxyguanosine triphosphate; dHMC-TP, hydroxymethyldeoxycytidine tri- 

 phosphate; dTTP, deoxythymidine triphosphate; DNA, deoxyribonucleic acid; HMC, 5- 

 hydroxymethylcytosine; UDPG, uridine diphosphate glucose; Tris, tris-(hydroxymethyl)amino- 

 methane; KPO4, potassium phosphate buffer; PRPP, 5-phosphoryl a-D-ribofuranose 1-pyro- 

 phosphate; E^, molar extinction coefficient. 



^ Lehman, I. R., M. J. Bessman, E. S. Simms, and A. Kornberg, /. Biol. Chem., 233, 163 

 (1958). 



^ Bessman, M. J., I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms, and A. Kornberg, 

 these Proceedings, 44, 633 (1958). 



^ Bessman, M. J., I. R. Lehman, E. S. Simms, and A. Kornberg, J. Biol. Chem., 233, 171 ( 1958); 

 Adler, J., 1. R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg, these Proceedings, 44, 

 641 (1958); Lehman, L R., S. B. Zimmerman, J. Adler, M. J. Bessman, E. S. Simms, and A. 

 Kornberg, these Proceedings, 44, 1191 (1958); Kornberg, A., Harvey Lectures, 53, 83 (1957- 

 1958). 



•6 Hershey, A. D., J. Dixon, and M. Chase, /. Gen. Physiol, 36, 777 (1952-1953). 



6 Wyatt, G. R., and S. S. Cohen, Biochem. J., 55, 774 (1953). 



^ Flaks, J. G., and S. S. Cohen, Biochim. et Biophys. Acta, 25, 667 (1957); Federation Proc, 

 17, 220(1958). 



8 Sinsheimer, R. L., Science, 120, 551 (1954); Volkin, E., /. Am. Chem. Soc, 76, 5892 (1954). 

 ' Sinsheimer, R. L., these Proceedings, 42, 502 (1956); Jesaitis, M. A., J. Exp. Med., 106, 

 233 (1957); Federation Proc, 17, 250 (1958). 



1° Streisinger, G., and J. Weigle, these Proceedings, 42, 504 (1956). 



11 Cohen, S. S., J. Biol. Chem., 174, 281 (1948). 



>' Kornberg, A., L R. Lehman, and E. S. Simms, Federation Proc, 15, 291 (1956), and more 

 recent unpublished observations. 



'^ An abstract of this work has appeared [Zimmerman, S. B., S. R. Kornberg, J. Josse, and A. 

 Kornberg, Federation Proc, 18, 359 (1959)], as have abstracts regarding an enzyme which phos- 

 phorylates dHMC-5-P (R. Somerville and G. R. Greenberg, Ibid., 327), dHMC-TP incorporation 

 into DNA, and a suggested dCTP-degrading enzyme (J. F. Koerner and M. S. Smith, Ibid., 264) 

 in T2-infected E. coli. 



i'* Anderson, E. H., these Proceedings, 32, 120 (1946). 



^^ We are indebted to Dr. Helen Van Vupakis for a generous gift of phage T2r "•". 



" Adams, M. H., in Methods in Medical Research, ed. J. H. Comroe, Jr. (Chicago: Yearbook 

 Publishers, Inc., 1950), 2, 1. 



1' Doermann, A. H., J. Gen. Physiol, 35, 645 (1952). 



'* When fluoride was present in the kinase assays, the nucleotides were adsorbed to and eluted 

 from Norit before phosphatase treatment in Stage II of the assay. 



" For large-scale enzyme preparations, 50-liter cultures at the late exponential phase (2 X 10" 

 cells/ml), in modified M-9 medium (see Methods) but lacking CaCL, were treated with 3-4 T2r"'' 

 per cell and 10 min later with 50 7 of chloramphenicol per ml. The culture was then chilled to 0° 

 with ice over a 10-min period and harvested by centrifugation. The initial steps in the purifi- 

 cation of the dHMC-5-P kinase, dCTPase, glucosylating enzyme, and hydroxy methylating 

 enzyme were the same. Sonic extracts, prepared in 5 volumes of 0.05 M glycylglycine buffer, 

 pH 7.0, were centrifuged and the supernatant fluid diluted with buffer to contain 10 mg of pro- 

 tein per ml. Streptomycin sulfate (5 per cent solution), equal to 0.3 volume of the diluted ex- 

 tract, was added; the supernatant fluid collected after centrifugation contained 3 to 4 mg of pro- 

 tein per ml. It was adjusted to pH 8 with KOH and applied to a diethylaminoethylcellulose 

 (Brown Co.) column [Peterson, E. A., and H. A. Sober, /. Am. Chem. Soc, 78, 751 (1956)], equi- 



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