I04 PETER REICHARD 



visualized to have at least a threefold purpose: (i) The synthesis of RNA; 

 (2) the synthesis of coenzymes; and (3) the synthesis of DNA: 



CO2, NH3, glucose, etc. 



I 



Ribonucleotides 



RNA Coenzymes Deoxyribonucleotides — >DNA 



In view of these considerations it seemed an attractive hypothesis to 

 consider the reductive step as a possible " pace maker " for DNA-synthesis, 

 and it was hoped that the construction of an /// vitro system carrying out 

 the synthesis of DNA from ribonucleotides might serve as a model system, 

 in which the influence of different factors on this hypothetical pace maker 

 might be studied. Ample evidence exists that such a first step in a reaction 

 sequence may indeed act as a rate-limiting step [6] and that it can be 

 influenced and regulated by the products of later reactions. 



In all experiments to be described here the source of enzyme was a 

 high-speed supernatant fraction from a homogenate of 5-day-old chick 

 embryos. Both cytidine and guanosine ribonucleotides were used as sub- 

 strates for DNA formation. 



First it was necessary to establish that our enzyme preparation could 

 synthesize DNA from labelled deoxynucleotides. It was found that in- 

 corporation of radioactivity from labelled dCMP or dGMP into DNA 

 required the presence of ATP, Mg + + and "primer" DNA. This in- 

 corporation was further stimulated by the addition of a complementary set 

 of the other deoxynucleoside triphosphates. These results demonstrate 

 that the chick embryo extract contained enzymes which catalyze the 

 formation of DNA from deoxyribonucleotides by a mechanism similar to 

 that described earlier for other systems [i, 7, 8]. 



Next the formation of radioactive DNA from labelled ribonucleotides 

 (CMP and GMP) was studied. It was found that this process again 

 required the addition of ATP, Mg + + and "primer" DNA. When either 

 ^-P-labelled or tritium( = base)-labelled CMP was used as substrate, it was 

 found that identical amounts of isotope were incorporated into DNA 

 (Fig. i). This experiment demonstrates that the intact nucleotide was 

 used for DNA synthesis and that the incorporation of isotope did not 

 occur as a result of, e.g., transglycosylation. 



It was then necessary to investigate in which manner the ribonucleo- 

 tide had been used for DNA synthesis. With techniques used earlier by 

 Adler et al. [9] it was possible to demonstrate that in our experiments the 

 isotopic ribonucleotide was first reduced to the deoxyribonucleotide, and 



