Metabolism and Energy Transformation in Body Cells - Renata Armenakovna Petrosova 2004

Template-Directed DNA Synthesis
DNA Replication

The self-duplication process of a DNA molecule, which ensures the precise copying of Genetic information, is called Replication or DNA duplication.

The Discovery of the replication mechanism was preceded by numerous experiments on DNA Synthesis. The DNA model proposed by James Watson and Francis Crick sparked a whole series of experiments to elucidate its replication mechanism. The most compelling evidence was presented in 1958 by scientists M. Meselson and F. Stahl. Three hypotheses were put forward during the course of the experiment.

1. Conservative replication. The double-stranded DNA molecule serves as a template for the synthesis of an entirely new DNA molecule; that is, the new DNA is an exact copy of the original.

2. Semiconservative replication. A second complementary strand is synthesized on each strand of the original DNA molecule. Each new DNA molecule consists of one original and one newly synthesized DNA strand.

3. Dispersive replication. The DNA molecule breaks down into short fragments that serve as templates for building the missing parts of new DNA molecules.

Escherichia coli, which contains only a single DNA molecule in its Cell, was used as the experimental Organism. To determine the correct version, the Bacteria were grown on a nutrient medium containing the radioactive nitrogen isotope ¹⁵N. After several generations, the DNA of all bacteria contained the ¹⁵N isotope (Fig. 24).

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Fig. 24. Hypotheses of the DNA replication process: 1. A — conservative; B — semiconservative; C — dispersive. II. Result of centrifugation of bacterial DNA molecules

Using centrifugation, these DNA molecules were isolated from the Cells as a distinct fraction, which proved that the DNA of all bacteria had the same isotopic composition. Next, the bacteria containing only the ¹⁵N isotope in their DNA were transferred to a nutrient medium with the common nitrogen isotope ¹⁴N. Centrifugation of the new generation of bacterial DNA showed that these molecules contained both ¹⁴N and ¹⁵N nitrogen, but they could not be separated into fractions by mass. Consequently, both isotopes were present in the same DNA molecule. The first hypothesis regarding the conservative mode of DNA replication was rejected because separate fractions of DNA with ¹⁴N and DNA with ¹⁵N were absent.

To test the remaining two hypotheses, the DNA of the third generation of bacteria was studied. It was successfully separated into two distinct fractions: DNA (¹⁴N) and DNA (¹⁴N, ¹⁵N). Thus, the third hypothesis of dispersive DNA replication was ruled out. The experiment established that DNA is synthesized in a semiconservative manner.

DNA replication is based on the following principles.

Complementarity. Each strand of a DNA molecule contains a nucleotide sequence that is precisely complementary to The nucleotide sequence on the opposite strand. Consequently, the new molecules carry the identical genetic information: strand A serves as a template for the synthesis of strand A', and strand A' serves as a template for the synthesis of strand A.

Semiconservative synthesis. If strands A and A' of a single DNA molecule are separated, each of them will serve as a template for the Synthesis of the corresponding missing strand. The new DNA molecules will contain one new strand and one original parental DNA strand. The two daughter DNA molecules are completely identical to the original parental molecule.

Antiparallelism. The two strands in a DNA molecule are antiparallel. This means that at the beginning of one strand, a nucleotide features a deoxyribose residue with a free hydroxyl group (-OH) at the 3' carbon atom, whereas at the beginning of the complementary strand, the nucleotide features a phosphoric acid residue linked to the 5' carbon atom of deoxyribose. Accordingly, the first strand ends with a nucleotide bearing a 5' end, and the second strand ends with a nucleotide bearing a 3' end.

Discontinuous (semi-discontinuous) synthesis. The enzyme DNA polymerase, which ensures the synthesis of a new DNA strand on the template of the original strand, joins NUCLEOTIDES exclusively in the 5'-to-3' direction. Therefore, synthesis proceeds continuously forward along one strand in the 5'–3' direction, and then backward along the other strand toward the 5' end in fragments. These individual fragments are joined together later.

The replication process proceeds in three stages.

Stage I — initiation. DNA replication begins with the unwinding of The Double Helix at one end; this process does not occur across the entire region of the molecule at once, but rather segmentally, in fragments. This reaction takes place in the presence of several protein factors. The two strands in a DNA molecule are bonded quite strongly, so special Proteins are required to separate them. Proteins of the first type move along the DNA molecule, untwisting the helix and breaking the Hydrogen Bonds between complementary bases, thereby unwinding the double helix. Proteins of the second type prevent the two strands from re-annealing and ensure the efficient action of the first-type proteins. In addition, they straighten single-stranded DNA and facilitate the progression of the DNA polymerase enzyme, which catalyzes the synthesis of new strands. As a result of the action of all protein factors, a Replication fork is formed (Fig. 25).

Fig. 25. DNA replication: I — Structure OF THE replication fork: 1 — DNA helicase (DNA-unwinding protein); 2 — single-strand DNA-binding protein; 3 — DNA polymerase; II — DNA synthesis on the leading and lagging strands (arrows indicate the direction of synthesis and movement of DNA polymerase)

Stage II — elongation. At this stage, actual DNA synthesis takes place. Nucleotides align along each DNA strand according to THE PRINCIPLE OF complementarity. The self-replicating enzyme DNA polymerase, moving along the replication fork, links nucleotides together in the 5'-to-3' direction. The enzyme moves in only one direction. Since the DNA strands are antiparallel, DNA polymerase moves in the opposite direction along the other DNA strand, synthesizing a fragment from the Water/144.html">Origin of the replication fork toward the end of the strand.

On the first strand, upon reaching the beginning of the replication fork, the enzyme effectively stimulates the movement of the binding protein. It shifts further up, unwinding the DNA molecule further. The enzyme moves along the first strand once again, continuing the interrupted synthesis from the last nucleotide. Meanwhile, on the opposite strand, the enzyme synthesizes a second DNA fragment in the reverse direction.

The replication fork turns out to be asymmetrical. One of the daughter DNA strands (the leading strand) is built continuously, while the other (the lagging strand) is synthesized discontinuously, in the form of separate fragments. These fragments are joined to each other later, only after the synthesis of subsequent fragments.

The final stage III is called termination. All protein factors and Enzymes are removed from the newly synthesized DNA molecules. The two daughter DNA molecules separate, coil into spirals, and acquire their proper structure. The DNA synthesis process comes to an end.

DNA copying occurs with high fidelity. On average, there is one error for every 1 · 10⁹ complementary nucleotide pairs formed during replication. These errors are corrected by a specialized proofreading system of proteins that recognize and remove incorrect nucleotide residues.

The accuracy of copying ensures the correct transmission of hereditary information. The entire replication process is driven by the energy of ATP.

Questions and Tasks for Self-Control

1. What hypotheses were proposed during The Study of DNA replication? Which method proved the semi-conservative principle of replication?

2. Explain The process of DNA replication. Why does the synthesis of the DNA molecule proceed fragmentarily rather than continuously along its entire length?

3. Which enzyme is involved in the synthesis of daughter DNA strands?

4. Determine The sequence of the second DNA strand if the first strand has the following nucleotide sequence: ЦТТААЦАЦЦГГГЦАТТЦЦГГГААТТГ.