I. REPLICATION OF DXA IN CELL- FREE SYSTEMS 51 



differences between the synthesis of DNA in the calf thymus system 

 and the less purified regenerating rat liver system described by Bollum 

 and Potter (1958) and Bollum (1958) are evident and therefore remarks 

 will be confined to the former. 



At the outset it may be stated that the reaction catalyzed by calf 

 thymus polymerase (purified about 50-fold) is very similar to the one 

 from E. coll. The requirement for all four deoxyribonucleoside triphos- 

 phates, jNIg ions, and DNA is evident in Table XXV. Omission of any 



TABLE XXV 



Requirements for Thymidine Triphosphate Incorporation 



WITH Calf Thymus Polymerase" 



HMTMP incorporated into DNA 

 Reaction mi.xture (MMmoles) 



Complete* 100 



Minus dATP 9 



Minus dGTP 13 



Minus dCTP 28 



Minus dATP, dGTP, dCTP 2 



Minus Mg++ 



Minus DNA 



" From Bollum (1960a). 



''The complete reaction mixture contained the following concentrations: deox>Tibo- 

 nucleoside triphosphates, 25 MHioles in each (H^-TTP containing 6700 cpm per m/xmole) ; 

 MgCl2, 10 mmoles; glycine buffer, pH 9, 25 mmoles; 25 jug DNA (salmon sperm); and 

 195 Mg of fraction C protein. Final volume, 0.20 ml. Incubated 20 minutes at 37°C. 



of these constituents reduces the rate of incorporation. The reaction is 

 inhibited by high concentrations of pyrophosphate and it can be shown 

 that DNA synthesis is reversible in this system by measuring the forma- 

 tion of deoxyribonucleoside triphosphates in the presence of polymerase, 

 DNA, Mg ions, and pyrophosphate. With the partially purified enzyme, 

 net synthesis of DNA can be demonstrated if heated DNA is used as the 

 primer. The products formed resemble the primer with respect to 

 adenine-cytosine or thymine-cytosine content as demonstrated in Table 

 XXVI. Again, the failure of native DNA to prime the reaction is demon- 

 strated in this table. 



This reduced priming capacity of native DNA was investigated by 

 Bollum (1959a,b) who showed that even at early stages in the purifica- 

 tion the calf thymus polymerase uses denatured DNA much more 

 efficiently than native DNA as primer. This is seen clearly in Fig. 17 

 where several heated and unheated DNA preparations are compared. 

 Except in the case of salmon sperm DNA, the unheated or native DNA 

 preparations are strikingly deficient as primers as compared to the heated 



