Organization and Replication of DNA in Vivo 



275 



thesis is at the ends of double strands in- 

 volves some of the following facts: When 

 any sample of natural or native, double- 

 stranded DNA is heated to an appropriate 

 temperature (near 98° C), the H bonds are 

 broken and the complementary strands sepa- 

 rate. Double-stranded DNA's with high 



A 4- T 



— ratios become single-stranded at a 

 G + C B 



lower temperature than do those with low 

 ratios. This result is expected since high- 

 ratio DNA is richer in A-T than low-ratio 

 DNA, each pair of which has one less H 

 bond than a C-G pair, so that less energy 

 is needed to break the smaller total of H 

 bonds. If the appropriately heated mixture 

 is cooled quickly, the chains remain single, 

 producing denatured DNA . That heat de- 

 naturation followed by quick cooling pro- 

 duces single strands from double helices can 

 be confirmed by the loss of that part of the 

 DNA X-ray diffraction pattern which de- 

 notes polystrandedness. The change to 

 single-strandedness is also accompanied by 

 an increase of as much as 40% in the ab- 

 sorption of ultraviolet light of 2600 A, so 

 that single-stranded DNA is relatively hy- 

 perchromic. It also is slightly denser than 

 double-stranded DNA. If the hot mixture, 

 containing denatured DNA, is cooled slowly, 

 base pairing occurs and renatured DNA is 

 obtained which shows a hypochromic effect 

 and, from X-ray diffraction studies, evidence 

 of double helices. 



The second test of endwise DNA synthesis 

 involves converting double-stranded, all-light 

 and all-heavy DNA to the single-stranded 

 condition and locating the positions of the 

 two types of single strands in the ultra- 

 centrifuge tube. The "hybrid" double- 

 stranded DNA is then made single-stranded 

 and is ultracentrifuged. This preparation 

 shows only two major components, one lo- 

 cated at the all-light single-stranded position 

 and the other at the all-heavy single-strand 

 position. This result also is inconsistent 



with the hypothesis being tested. Not only 

 do the two tests eliminate the view that ap- 

 preciable endwise synthesis of DNA occurs 

 in bacterial DNA, but they offer additional 

 support for the hypothesis of replication 

 after strand separation. 



Similar experiments yielding similar re- 

 sults have been performed using the uni- 

 cellular plant, Chlamydomonas, and higher- 

 organisms, including man. The general 

 agreement in the results of all these experi- 

 ments apparently furnishes conclusive proof 

 of the correctness of the Watson-Crick hy- 

 potheses for the double-helix configuration 

 of chromosomal DNA and for its replication 

 after strand separation. 



Although the nuclear DNA of most or- 

 ganisms is present as nucleoprotein, being 

 combined with histones or protamines, the 

 DNA in bacteria and in the viruses attack- 

 ing them seems to exist uncombined with 

 basic protein. • In the latter case, the DNA- 

 containing fibers have a diameter of about 

 25 A. Ordinary chromosomes are probably 

 polynemic with respect to DNA double 

 helices, 4 although the exact number of double 

 helices per chromatid is not yet known with 

 certainty. The basic fibril in protamine- 

 containing sperm seems to be about 40 A 

 in diameter, containing one DNA double 

 helix plus basic protein. 3 In cells contain- 

 ing histones, two DNA double helices bound 

 side by side, plus the histone. form a fibril 

 which is about 100 A thick.' 1 



So far, the evidence presents no clue as 

 to how either end of a DNA polymer termi- 

 nates. The possibility exists that DNA is 

 a circular molecule, although this explana- 

 tion still leaves the problem of how chain 

 separation occurs if there are no free ends 

 to revolve. When DNA is extracted from 

 human sperm, about 0.1% of the "purified" 

 material is reported to be composed of amino 



3 See H. Ris and B. L. Chandler (1964). 



4 See W. J. Peacock (1963). 



