I. KEPLICATION OF DNA IN CELL-FREE SYSTEMS 55 



been reviewed by Cohen (1961). Many of the:<e new activitie.s exj)lain 

 several observations concerning the DNA metabolism of the infected cell. 

 For example, the induction of a specific enzyme which catalyzes the 

 hydroxymethylation of deoxycytidylate (Flaks and Cohen, 1957, 1959a) 

 accounts for the presence of this unique pyrimidine base in bacteriophage 

 DNA. Also, the marked increase in activity of a dCTPase (Zinnnerman 

 and Romberg, 1961; Koerner et al., 1960) may account for the apparent 

 decrease in dCMP kinase activity in extracts of bacteriophage-infected 

 cells. Romberg et al. (1959), in a study of the effect of bacteriophage 

 infection on DNA synthesis, observed a 10-fold stimulation of poly- 

 merase activity in extracts of T2-infected cells but noticed that the 

 observed increase was dependent on the type of primer used for the 

 assay. That is, the increased activity after infection could only be dem- 

 onstrated if heated DNA was used as primer. Recently, Aposhian and 

 Romberg (1961, 1962) have conducted a careful study of the polymerase 

 in extracts of infected cells and provide conclusive evidence that the 

 increased activity after infection is due to the formation of a new 

 polymerase. 



Polymerase from T2-infected cells was purified 600-fold and was 

 distinguishable from E. coli polymerase in the following respects: 



1. Levels of antiserum against E. coli polymerase causing 100% 

 inhibition have no effect on T2 polymerase and vice versa. 



2. 7;-Chloromercuribenzoate (1.7X10"^^^^) inhibits T2 polymerase 

 completely whereas E. coli polymerase is inhibited 27%. 



3. T2 polymerase and E. coli polymerase are easily separable by 

 chromatography on a column of phosphocellulose. 



4. The two polymerases have distinctly different primer requirements. 

 Except for this last property the differences between the two enzymes 



should have no consequences for the in vitro synthesis of DNA. Thus, 

 the T2 enzyme is similar to the E. coli enzyme in that it requires all four 

 deoxyribonucleoside triphosphates, Mg ions, and DNA. It can also 

 incorporate base analogs into DNA in the same manner as the E. coli 

 enzyme provided the analogs are supplied as the deoxyribonucleoside 

 triphosphates. 



The most striking property of the T2 enzyme is its characteristic 

 dependence on heated DNA first observed in crude extracts of infected 

 cells. The E. coli enzyme and T2 polymerase are compared in Table 

 XXVII; it may be seen that heated DNA is a better primer for the 

 T2 polymerase-catalyzed reaction but is actually somewhat worse for 

 the E. coli polymerase reaction. That this difference in behavior is not 

 due to some unknown factor in either the purified T2 or E. coli polymer- 

 ases is shown by the mixture of the two preparations. In this case the 

 values equal the sums of the individual incubations. 



