io6 



PETER REICHARD 



of DNA, the isotope stays attached to the nucleotide used as substrate for 

 the DNA polymerase. 



(b) With micrococcal DNA-se + spleen diesterase. This results in 

 the formation of 3 '-nucleotides. ^^P is no longer attached to the nucleotide 

 used as substrate, but is transferred to the neighbour nucleotide. 



The results of Fig. 2 obtained by degradation according to (a) show 

 that CMP was used for DNA synthesis after transformation to dCMP, 

 since about 95° o of the isotope resides with this deoxyribonucleotide. 

 After degradation according to {b) the isotope is distributed among all 

 four deoxyribonucleotides. This in turn demonstrates that labelled dCMP 

 (formed from CMP) in DNA was linked to all four deoxynucleotides in 

 internucleotide linkage. 



Fig. 3. Stimulation of DNA formation from dCMP by an ATP-regenerating 

 system. Conditions as in Fig. i (ooii fxmole of dCMP substituted for CMP). 

 Where indicated 4 ■ 5 /xmoles of creatine phosphate + o • i mg. of creatine kinase 

 were added. 



The results obtained so far strongly indicate that the chick embryo 

 enzyme preparation carried out the synthesis of DNA from ribonucleo- 

 tides according to the following general reaction sequence : 



Ribonucleotide- — ^Deoxyribonucleotide >DNA 



It was now possible to study some of the factors which might influence 

 this overall pathway of DNA synthesis. 



The first question studied was how the process was influenced by the 

 ATP-level in the system. It might be expected — and has been shown 

 already for other similar systems — that a high level of triphosphates, as is 

 obtained by the addition of an ATP-regenerating system, is favourable for 

 the synthesis of DNA from a deoxyribonucleotide. Figure 3 demonstrates 

 that the addition of creatine phosphate + kinase stimulated isotope in- 

 corporation from labelled dCMP into DNA, and that the chick embryo 

 system thus conformed to expectation. 



However, DNA formation from a ribonucleotide, such as CMP, was 

 decreased when an ATP regenerating system was added (Fig. 4). The 



