322 



CHAPTER 35 



shown by its release from the precipitated 

 DNA when this was treated with DNAase. 



Although this result is not impressive 

 quantitatively, it furnished C'Mhymidine, 

 incorporated into acid-precipitable, DNAase- 

 sensitive material; the amount formed, of 

 this labeled material, could be used as an 

 assay of the effect of changes in the experi- 

 mental procedure. Let us see how this led to 

 a change in the procedure and to a better 

 understanding of the nature of the reaction. 



Derivatives of adenosine are commonly 

 formed from reactions involving ATP; deriv- 

 atives of uridine, cytidine, and guanosine 

 hkewise involve their respective triphosphates 

 and the liberation of inorganic pyrophos- 

 phate. Such facts lead us to conclude that 

 the basic building block, in the formation of 

 diribotides or polyribotides, is the riboside 

 5'-phosphate, when activated in the form of 

 the riboside 5'-triphosphate. It is a reason- 

 able hypothesis, therefore, to suppose that 

 the active building block of polydeoxyribo- 

 tides is the deoxyriboside 5'-triphosphate. 



If this is so, the ATP added to the extract 

 of the in vitro experiments might be function- 

 ing to convert deoxyribosides to the 5'- 

 triphosphate condition (making specifically 

 C'Mhymidine 5'-triphosphate). This view 

 was supported when DNA synthesis was ob- 

 tained in vitro using labeled thymidine 5'- 

 triphosphate (T*PPP), instead of labeled 

 thymidine (T*) + ATP (ARPPP). 



In order to learn more about the ingredi- 

 ents essential for DNA synthesis, the initial 

 extract, obtained from the sonic treatment of 

 bacteria, was fractionated and its protein 

 concentrated. This was accompanied by 

 about a 4,000-fold increase in synthetic 

 activity. From this and other evidence, it 

 became clear that the presence of a protein 

 catalyst, or enzyme, was essential for the 

 synthetic reaction to proceed. 



Once the enzyme was concentrated, it was 

 possible to obtain a large net increase (final 

 amount minus initial amount) in DNA. 



However, such a net increase was obtained 

 only if the 5'-triphosphates of all four de- 

 oxyribosides commonly found in DNA were 

 added to in the incubation mixture. De- 

 oxyriboside S'-fi'/phosphates were not active, 

 nor were riboside 5'-triphosphates. The other 

 requirements, for net increase in DNA 

 amount, were the presence of already formed 

 DNA of high molecular weight, Mg ions, and 

 the enzyme-containing protein. The already 

 formed, high molecular weight DNA, which 

 serves as a primer for the reaction, may come 

 from a plant, animal, bacterial, or viral 

 source. Similar DNA synthesizing extracts 

 can be prepared from other bacteria and 

 various animal tissues. 



Using E. coli preparations, it is possible to 

 obtain an extended synthesis of DNA which 

 produces 20 or more times as much DNA as 

 was present at the start (that is, than there was 

 primer). In this case, therefore, 95% or more 

 of the DNA present at the end must have 

 been synthesized from the triphosphates 

 added as substrate. The extended synthetic 

 reaction proceeds until the supply of one of 

 the four triphosphates is exhausted, and re- 

 leases one inorganic pyrophosphate for each 

 deoxyribotide incorporated into DNA. 



While there is no extended synthesis of 

 DNA, if only one of the deoxyriboside 5'- 

 triphosphates is added as substrate, there is 

 some incorporation of this nucleotide into 

 the DNA chain in what is called a limited 

 reaction. By what mechanism does the 

 nucleotide add on to the DNA chain? Sup- 

 pose the only triphosphate added to the sub- 

 strate was deoxycytidine 5'-triphosphate, 

 whose innermost phosphate carried radio- 

 active P*- (6fCP*PP). The two possible ways 

 the DNA chain might lengthen are shown at 

 the left and right of Figure 35-3. The DNA 

 chain which is present as primer is indicated 

 there by being enclosed in brackets. The 

 primer chain can be considered to have a 

 nucleotide end (top) (to which pyrophosphate, 

 P-P, is shown added in the diagram to the 



