Physical properties of enzymatically synthesized DNA. 



The first line of evidence is derived from studies of the physical nature of 

 the DNA produced by the enzyme. It may be mentioned again that in these 

 descriptions as in those of the chemical nature of DNA, to be discussed shortly, 

 90 — 95 % of the DNA sample comes from the substrates used in the reaction. 

 From collaborative studies with Dr. Howard K. Schachman, to whom we 

 are greatly indebted, it can be said that the enzymatic product is indistinguish- 

 able from high-molecular weight, double-stranded DNA isolated from nature 

 (17). It has sedimentation coefficients in the neighbourhood of 25, reduced 

 viscosities of 40 deciliters per gram and, on the basis of these measurements, 

 it is believed to be a long, stiff rod with a molecular weight of about 6 million. 

 Upon heating the DNA, the rod collapses an'd the molecule becomes a compact, 

 randomly coiled structure; it may be inferred that the hydrogen bonds holding 

 the strands together have melted and this is borne out by characteristic 

 changes in the viscometric and optical properties of the molecule. Similar 

 results are found upon cleavage of the molecule by pancreatic deoxyribo- 

 nuclease. In all these respects the enzymatically synthesized DNA is indis- 

 tinguishable from the material isolated from nature, and may thus be presumed 

 to have a hydrogen-bonded structure similar to that possessed by natural DNA. 



Would one imagine that the collapsed jumbled strands of heated DNA 

 would serve as a primer for DNA synthesis? Very likely one would think not. 

 Guided by intuition derived from everyday experience with a jumbled strand 

 of twine one might regard this as a hopeless template for replication. It turns 

 out that the collapsed DNA is an excellent primer and the nonviscous, ran- 

 domly coiled, single-stranded DNA leads to the synthesis of highly viscous, 

 double-stranded DNA (18). Sinsheimer has isolated from the tiny 0X 174 

 virus a DNA which appears to be single-stranded (19). Like heated DNA it 

 has proved to be an excellent primer (18) and a favorable material in current 

 studies (20) for demonstrating in density gradient sedimentations that it is 

 progressively converted to a double-stranded condition during the course of 

 enzymatic synthesis. 



While a detailed discussion of the physical aspects of replication is not 

 feasible in this lecture, it should be mentioned that the DNA in the single- 

 stranded condition is not only a suitable primer but is the only active form 

 when the most purified enzyme preparations are used. With such coli prepa- 

 rations, the native, double-stranded DNA is inert unless it is heated or pre- 

 treated very slightly with deoxyribonuclease. Bollum has made similar ob- 

 servations with the enzyme that he has uurified from calf thymus (21). 



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