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Chapter *35 



REPLICATION OF DNA 

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ELF-REPLICATION was assumed, 

 |On p. 9, to be a characteristic 

 of the genetic material. In view 

 of the indirect evidence that the DNA normal- 

 ly conserved in chromosomes is genetic ma- 

 terial, we are particularly interested in learn- 

 ing more about the double helix of DNA and 

 how it accomplishes its replication after chain 

 separation. Although we can accept the 

 evidence (in Chapter 34) as being fairly con- 

 clusive that complementary chains are synthe- 

 sized after chain separation, no evidence was 

 presented bearing on the mechanism by 

 which this replication is accomplished. Fig- 

 ure 34-6 and the discussion on page 312 only 

 postulate a mechanism, which includes an 

 enzyme that joins the nucleotides together so 

 as to complete the new complementary strand. 

 Since the linear combination of nucleotides 

 doubtless requires energy, we should con- 

 sider the source from which this energy might 

 be derived. There is abundant evidence that 

 considerable chemical energy is contained in 

 the ribonucleotide adenosine triphosphate 

 {ATP) which is a riboside 5'-triphosphate 

 (Figure 35-1). ATP is known to react with 

 (1) nicotinamide mononucleotide (a ribonu- 

 cleotide, or ribotide) to produce a combina- 

 tion of the two, with the resultant elimination 

 of two phosphates as inorganic pyrophosphate 

 (Figure 35-2a), and (2) flavin mononucleo- 

 tide to produce a dinucleotide plus inorganic 

 pyrophosphate (Figure 35-2b). Evidence 

 that it is ATP which contributes the 

 pyrophosphate, by losing its two terminal 

 320 



OH OH 



FIGURE 35-1. 



Adenosine 5'-triphosphate {ATP) {ARPPP). 



phosphates, is obtained from the reaction of 

 ATP with certain acids (Figure 35-2c). Since 

 ATP supplies the energy for many chemical 

 reactions in the cell, it is reasonable to suppose 

 that it may also supply the energy to join the 

 separate deoxyribonuckotides {deoxyribotides) 

 into a DNA strand during replication. 



In view of the fact that DNA can be re- 

 moved from the nucleus, and can be separated 

 from protein, and still retain what appears 

 to be the main characteristics which it had 

 before it was removed (i.e., it presumably 

 retains the properties it had in the living cell), 

 we can hope to study DNA synthesis under 

 nonliving conditions. What should we ex- 

 tract from cells in order to study DNA 

 synthesis in vitro? Initially, we ought to 

 make use of all the apparatus that the cell 

 normally provides for this function. On the 

 chain separation view, DNA is needed to 

 serve as a template for new DNA synthesis, 

 so our extract should contain the DNA of the 

 cell. We should add ATP to the extract as a 

 source of the energy required for the synthe- 

 sis; we can also add magnesium ions, in the 

 form of MgCl2, for this serves to activate 

 many enzymes, including perhaps one which 

 might be required for DNA chain formation. 



How will we be able to tell whether DNA 

 was synthesized in the extract? Any crude 

 extract from cells might be expected to con- 

 tain DNAases which might depolymerize or 

 Otherwise degrade DNA as fast or faster than 



