780 



BIOCHEMISTRY: KORNBERG ET AL. 



Proc. N. a. S. 



The C '"-glucose fixed in DNA was rendered acid-soluble by crystalline pan- 

 creatic deoxyribonuclease. When 52 per cent of the nucleotides were no longer 

 acid-precipitable, 88 per cent of the glucose had been made acid-soluble. When the 

 C'-glucosylated HMC-DNA was digested to completion under conditions which 

 Lehman has found^^ to yield a quantitative conversion of phage T2 DNA to 5'- 

 mononucleotides, over 95 per cent of the radioactivity was found in the HMC 

 deoxynucleotide fractions of the ion-exchange chromatogram. 



Increase Rate of DNA Synthesis upon Infection.— DNA synthesis, measured by 

 the standard assay, ^ but with dHMC-TP instead of dCTP, is increased 12-fold in 

 extracts prepared 19 min after infection with T2 (Fig. 6). Little or no DNA syn- 

 thesis can be measured in these extracts when dCTP replaces dHMCTP. However 



o 



la 

 o. 



d« 



E 



16 



12 



E 



0- 



Fig. 6 — DNA "polymerase" levels before 

 and after infection with phage T2 with 

 dHMC-TP as substrate in place of dCTP. 

 The arrow indicates the start of infection 

 ("zero minutes," see Method I). The incu- 

 bation mixtures (0.30 ml) contained: 10 

 niMmoles each of dGTP, dATP, dTTP and 

 dHxMC-TP (C'S 1 X 103 cpm per m/imole), 

 Tris buffer, pH 7.5 (0.07 M), MgCU (0.007 

 M), 2-mercaptoethanol (0.001 M), 0.04 ml. 

 of "heated DNA" and sonic extract con- 

 taining 10-50 fig of protein. The "heated 

 DNA" was prepared by heating at 100° 

 for 5 min, at pH 9.2, an extract of T2- 

 infected cells; this preparation could be 

 purified without loss of activity by treat- 

 ment with ribonuclease, Norit, dialysis and 

 precipitation with acid; it was inactivated 

 by treatment with deoxyribonuclease. Fur- 

 ther details of procedure were as referred 

 to in Methods. 



TlME-minutes 



using 0.005 M F^ to inhibit dCTPase, DNA synthesis was elevated to levels near 

 those observed in assays with dHMC-TP. 



It should be emphasized that these measurements of rates of DNA synthesis 

 with dHMC-TP were made with heated DNA as primer; when unheated DNA was 

 used, there was no demonstrable increase in rate in the infected cell extracts. 

 Heated DNA of phage T2 or calf thymus origin served as well as that used in Figure 

 6. The basis for this requirement for heated DNA with infected cell extracts 

 requires further investigation. 



DISCUSSION 



In addition to the interest inherent in understanding the nature of viral infection 

 of a cell, studies of the pathway of DNA synthesis in infected cells provide a means 

 of testing and expanding our conceptions about the mechanism of DNA replication 

 in normal cells. 



133 



