The biosynthesis of the DNA must consist in a replication or duplication. The study of the DNA structure as proposed by Crick and Watson (see fig. 6-15) gives an idea of the mechanism of this replication. Each nucleotide of the template DNA chain need only be recognized by a complementary nucleotide before the latter is incorporated at the end of the chain in formation.
To this end, the double helix must therefore be open, at least partly and transitorily, to permit the pairing of the 2 complementary nucleotides by hydrogen bonding. The nucleotide thus selected is then incorporated in the chain being synthesized and 2 molecules identical to the starting molecule will be finally obtained (see fig. 6-26).
1. The Replication is Semi-Conservative:
In fact, two mechanisms of replication can be imagined (fig. 6-27):
i. The conservative mechanism, where one of the strands of the original molecule serves as template for the synthesis of a complementary strand; this newly formed strand serves in its turn as template for the synthesis of a strand which is complementary to it. Therefore, after a replication cycle, the starting molecule remains intact (hence the name of this mechanism) and a new molecule consisting of 2 newly synthesized strands is formed;
ii. The semi-conservative mechanism, thus named because each strand of the original molecule serves as template for the synthesis of a complementary strand, with the result that after a duplication cycle, there are 2 molecules of hybrid DNA, consisting of one strand of the original molecule paired with a newly formed strand.
The experiments of Meselson and Stahl could show that DNA replication takes place according to the semi-conservative mechanism. They cultivated E.coli for several generations in a mixture containing 15NH4Cl as the only source of nitrogen, with the result that the DNA synthesized was heavy.
At a given time (time 0), they transferred the culture to a mixture containing 14NH4Cl. Then, at regular intervals, they analyzed by centrifugation in cesium chloride gradient, the DNA extracted from the bacteria. This technique permits the separation of molecules as a function of their density; therefore, in the experiment described here, the denser the DNA, the more it will migrate to the bottom of the tube.
Meselson and Stahl obtained the following results:
i. At Time 0:
A single band, corresponding to the heavy DNA;
ii. After One Generation in the Mixture Containing 14N:
A single band having a density intermediate between the density of the heavy DNA and that of the light DNA, which means that the DNA molecules consist of an original heavy strand (15N) and a newly synthesized light strand (14N);
iii. After 2 Generations in the Mixture Containing 14N:
Two bands of equal intensity, one corresponding to the hybrids (heavy-light), the other corresponding to the molecules consisting of two light strands.
These results are in conformity with those expected in the case of a semi- conservative replication, as may be observed in figure 6-27, which also indicates the results that would have been observed if the replication had taken place in the conservative mechanism (it is noted that in this case one would never observe the formation of hybrids of intermediate density).
Experimental results therefore exclude the conservative mechanism (see fig. 6-27).
2. The Polymerization of Nucleotides is Carried Out by an Enzyme:
Although Watson and Crick had suggested in 1953, that the precursors of deoxyribonucleic acids can pair and bind one another without any enzymatic action, in 1958 an enzyme capable of polymerizing the 2′-deoxy- ribonucleosides-5′-triphosphates was isolated, by Kornberg from E.coli (DNA polymerase I). Subsequently, DNA polymerases were found in all bacteria and extracts of animal or plant cells in which a DNA synthesis could be observed.
3. The DNA Polymerase needs a DNA Template and a Primer:
The substrates of polymerization are compulsorily 2′-deoxyribonucleosides- 5’triphosphates (dNTP). As shown by figure 6-28, the DNA polymerase catalyzes the lengthening of polydeoxyribonucleotide chains (the term elongation is generally used) in the direction 5′ → 3′.
It must be noted that:
(1) The phosphodiester bond forms between the 3′ OH group of the growing chain and the 5′-phosphate group of the nucleotide to be incorporated. The growing chain is called primer;
(2) Each new deoxyribonucleotide to be incorporated is selected as complementary to the nucleotide situated just opposite, on the chain to be copied. This chain is therefore called template.
Therefore, no known DNA polymerase can synthesize DNA without a primer and a template, hence the exact name of this enzyme: DNA-dependent-DNA polymerase. Moreover, the DNA polymerases cannot synthesize the primer; the latter must be provided to them, hence the importance of priming which we will discuss again.
4. Polymerization Takes Place Only in One Direction: 5’→ 3′:
A DNA polymerase catalyzes the following reaction (see fig. 6-28):
The enzyme has two substrates. One is the couple template-primer 3’OH. The other substrate is compulsorily a deoxyribonucleosides-5′-triphosphate.
Some points could be confirmed by studying the nearest neighbour’s frequency. This is done by introducing in the polymerization reaction, the dNTP, one of them labeled with 32P on the a phosphate group (the one bound to carbon 5′ of pentose). At the end of the reaction, the DNA formed is subjected to an enzymatic hydrolysis by micrococcal DNase and phosphodiesterase.
In this way, the phosphodiester bonds are specifically split between carbon 5′ of deoxyribose and phosphate, and the deoxy- ribonucleosides-3′-monophosphates are liberated quantitatively.
Hence, as may be seen in figure 6-29, the 32P which was incorporated in the DNA when it was bound in position 5′ of a nucleoside-triphosphate, is found, after hydrolysis, attached in position 3′ of the adjacent nucleotide (i.e., the 32P has shifted from the nucleotide which carried it, to its nearest neighbour in the chain).
Separating the 4 deoxyribonucleosides-3′-monophosphates and determining their radioactivity, one can calculate the frequency at which each base was adjacent to a given base.
This type of study has led to three important conclusions:
i. The frequencies of the nearest neighbours of the template DNA and newly synthesized DNA are identical, which confirms that the DNA added to the reaction does serve as a template;
ii. Any sequence of the DNA template is copied, without preference nor specificity;
iii. The polarity of the newly synthesized chain is the opposite of that of the template chain. The growing chain and the template chain are also said to be “anti-parallel”. This conclusion is based on the fact that the equal frequencies of dinucleotide sequences which may be expected in the case of opposite polarities are verified by experiments, while those expected in the case of identical polarity of the 2 strands are contradicted by experimental results (see fig. 6-30).
