96 J. N. DAVIDSON 



(iii) The polymerization of the triphosphates in the presence of DNA 

 primer to yield new deoxyribopolynucleotide under the influence 

 of the polymerase enzyme. 



The enzymes involved in these reactions are freely soluble in water or 

 bufi^er solutions and are present in extracts obtained by centrifuging dis- 

 rupted cells at high speeds to remove particulate material. The overall 

 reaction involved in the combination of the second and third steps men- 

 tioned above can readily be demonstrated by incubating such extracts 

 with labelled thymidine (TdR) and following its incorporation into DNA 

 (Fig. i). Thymidine has several advantages for this purpose: it is a specific 

 precursor of DNA and it can readily be obtained commercially, labelled 

 with tritium at high levels of activity. For these reasons it has been ex- 

 tensively employed in autoradiographic studies on DNA biosynthesis, 



TdR 



thymidine Jcjn<3s<z 



TMP^-dUMP—UMP 



TMP k/n<3s<z 

 TDR 



TDP Ji/nasiz 

 TTP dATP dGTP dCTP 



po/ym<zrsis<z 



DNA 



Fig. I. Pathway of incorporation of thymidine into DNA. 



especially as tritium is a label peculiarly suitable for autoradiography (for 

 bibliography see ref. [18]). But thymidine has the further advantage, as 

 will be mentioned later, that the enzymes involved in its phosphorylation 

 to its triphosphate (TTP) increase and decrease according to the state of 

 proliferation of the tissue whereas those involved in the phosphorylation 

 of the deoxyribonucleoside monophosphates of adenine, guanine and 

 cytosine show a much smaller, if any, variation [13, 14]. 



Against these advantages it might be argued that thymidine is not a 

 normal metabolite on the anabolic pathway to TTP in vivo since the 

 formation of thymine derivatives proceeds at the nucleotide level by way 

 of UMP and dUMP to TMP (Fig. i), but this is not a serious disadvantage 

 since the kinase involved in the phosphorylation of thymidine to TMP 

 appears to run parallel with the kinases phosphorylating TMP to TTP [13]. 



The incorporation of pH]-TdR into DNA by the enzyme systems in 

 cell-free extracts of several tissues is illustrated in Table L As might be 



