NH, 



Chapter 21 



REPLICATION OF DNA 

 IN VITRO 



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O— P— O— P— o— p— o- 



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Iarly in this book (p. 10) we 

 assumed self-replication to be 

 a characteristic of the genetic 

 material. In light of the indirect evidence 

 that chromosomal DNA is genetic material, 

 it is of great interest to learn as much as 

 possible about the replication of the DNA 

 double helix. Although the evidence (Chap- 

 ter 20) is fairly conclusive that comple- 

 mentary chains are synthesized after chain 

 separation, no evidence has yet been pre- 

 sented about how this replication is accom- 

 plished. Figure 20-7 and the discussion on 

 page 271 only postulate a mechanism which 

 includes an enzyme that joins the nucleotides 

 forming a new complementary strand. 



Since the linear combination of nucleo- 

 tides undoubtedly requires energy, consider 

 the possible source of this energy. Consid- 

 erable chemical energy is contained in the 

 ribotide, adenosine triphosphate (ATP), a 

 riboside 5 '-triphosphate (Figure 21-1). 

 The energy hitherto needed to bond two 

 phosphates to adenosine monophosphate be- 

 comes available when ATP reacts with other 

 nucleotides or acids and loses its two ter- 

 minal phosphates as inorganic pyrophos- 

 phate. Because ATP is known to supply 

 the energy for many chemical reactions in 

 the cell, it is reasonable to suppose that it 

 may also supply the energy needed to join 

 individual deoxyribotides to a DNA strand 

 during replication. 



Since DNA removed from the nucleus and 

 separated from protein still retains what ap- 

 279 



OH OH 



FIGURE 21-1. 



Adenosine 5' -triphosphate (ATP) (APPP). 



pear to be its main characteristics in situ 

 (in the living cell), we may well be able to 

 study DNA synthesis under nonliving condi- 

 tions. What should we extract from cells 

 in order to study DNA synthesis in vitro? 

 Basically, we ought to use all the apparatus 

 the cell normally utilizes for this function. 

 From the strand separation viewpoint, DNA 

 is needed to serve as a template for DNA 

 synthesis, so the extract should contain the 

 cell's DNA. ATP is added to the extract 

 as the source of energy required for the 

 synthesis. MgCl. can also be added; since 

 the magnesium ion, Mg++, is known to 

 activate many enzymes, perhaps it will also 

 act on the one required for DNA strand 

 formation. 



How can we tell whether DNA is syn- 

 thesized in the extract? Any crude cellular 

 extract would be expected to contain DNases. 

 These enzymes might depolymerize or other- 

 wise degrade DNA as fast as — or faster 

 than — any process synthesizing DNA. The 

 problem of identifying DNA synthesis in the 

 absence of a net increase in DNA quantity 

 can be solved by preparing the deoxyribo- 

 side thymidine with radioactive C u incor- 

 porated in its pyrimidine and adding this 

 •'hof chemical to the extract. If any radio- 

 actively-labeled thymidine is incorporated 

 into DNA, it would happen as part of the 

 synthetic reaction, since incorporation into 

 DNA only occurs during synthesis. 



