280 



CHAPTER 21 



Finally, we ought to obtain the extract 

 From cells that are growing and dividing 

 rapidly, for these cells are likely to contain 

 the greatest amount of functional apparatus 

 for DNA synthesis. In line with this rea- 

 soning, an experiment is performed with an 

 extract of the bacterium Escherichia coli? 

 ATP, Mg " ions, and radioactive thymidine 

 are added and the pH is adjusted to suit 

 experimental conditions. After an incuba- 

 tion interval (about 30 minutes), the pH 

 is made suitably acidic for precipitating a 

 DNA polymer; single deoxyribosides — that 

 is, monomers — remain soluble. The acid 

 precipitate is washed many times until it is 

 certain that the DNA precipitate is not 

 contaminated by adsorbed deoxyribosides. 

 When the DNA is examined, it is found to 

 be only slightly radioactive (50 counts per 

 unit time as compared with 5 million counts 

 in the thymidine substrate added). In fact, 

 so little thymidine is incorporated in the 

 DNA that it is 10,000 times too small to 

 be detected by ordinary chemical analysis. 

 Nevertheless, the radioactivity is without 

 doubt due to thymidine incorporated into 

 DNA and can be released from the precipi- 

 tated DNA by treatment with DNase. 



Although this result is not quantitatively 

 impressive, the process furnishes C ,4 -thymi- 

 dine-labeled, acid-precipitable, DNase-sensi- 

 tive DNA as the end product. The amount 

 of this labeled material formed can be used 

 to determine the effect of changes in the ex- 

 perimental procedure. This fact has already 

 led to a change in the procedure and to a 

 better understanding of the nature of the 

 reaction. 



Reactions that produce derivatives of 

 adenosine commonly start with ATP as one 

 of the reactants. Similarly, derivatives of 

 uridine, cytidine, and guanosine involve their 

 respective triphosphates and the liberation 



1 The preceding and following account is based 

 primarily upon work by A. Kornberg and his 

 associates. 



oi inorganic pyrophosphate. Such facts lead 

 to the conclusion that the fundamental unit 

 in the formation of diribotides or polyribo- 

 tides is the riboside 5'-phosphate, activated 

 in the form of riboside 5 '-triphosphate. It 

 is reasonable, therefore, to assume that the 

 active building block of polydeoxyribotides 

 is the deoxyriboside 5'-triphosphate. 



If this molecule is the building block, the 

 ATP added in the in vitro experiments may 

 be converting various deoxyribosides — al- 

 ready present or added to the extract — to 

 the 5 '-triphosphate condition (making, for 

 instance, C 1 '-thymidine 5'-triphosphate). 

 This view is supported since DNA synthesis 

 occurs in vitro when labeled thymidine 5'- 

 triphosphate (T*PPP) is used instead of 

 labeled thymidine (T*)+ATP (APPP). 



To learn more about the ingredients es- 

 sential to DNA synthesis, the initial extract, 

 obtained from the sonic treatment of bac- 

 teria, is fractionated and its protein, concen- 

 trated. This procedure results in a nearly 

 4,000-fold increase in synthetic activity. 

 From this and other evidence, it becomes 

 clear that the presence of a protein catalyst — 

 the enzyme E. coli DNA polymerase (or 

 DNA duplicate) — is essential for the syn- 

 thetic reaction to take place. 



Once E. coli DNA polymerase is concen- 

 trated, it is possible to obtain a large net 

 increase in DNA (final amount minus initial 

 amount). Such a net increase, however, is 

 obtained only if the 5'-triphosphates of all 

 four deoxyribosides commonly found in 

 DNA are added to the incubation mixture. 

 Deoxyriboside 5'<7/phosphates are not active, 

 nor are riboside 5'-triphosphates. The other 

 requirements for net increase in DNA 

 amount are: 



1. The presence of already-formed DNA 

 molecules of high molecular weight 



2. Mg+ f ions 



3. DNA polymerase. 



The already-formed, high molecular weight 



