Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND REALIZATION OF GENETIC INFORMATION

CHAPTER 20. MOLECULAR MECHANISMS OF DNA REPLICATION AND RNA TRANSCRIPTION

20.1. BIOLOGICAL SIGNIFICANCE OF DNA REPLICATION. SEMICONSERVATIVE MECHANISM OF REPLICATION

The Significance of the Discovery of the DNA double helix Structure by J. Watson and F. Crick in 1953 went far beyond deciphering the geometrical STRUCTURE OF THE molecule. The proposed model opened up possibilities for establishing clear scientific concepts regarding The Mechanism of duplication—Replication of the DNA molecule, that is, the equal distribution of genetic material between daughter Cells, which was arguably the greatest biological mystery throughout human history.

Class="center">

Fig. 20.1. James D. Watson (born 1928), American biochemist, one of the founders of molecular biology. Nobel Prize (1962).

Fig. 20.2. Francis Crick (born 1916), English physicist, one of the founders of molecular biology. Nobel Prize (1962).

As J. Watson later noted, "the existence of two intertwined chains with identical base sequences could not be accidental. On the contrary, this gives reason to believe that one of the chains of each molecule serves at a certain stage as a template for the Synthesis of the other chain. According to this scheme, Gene replication would begin with its Separation into two identical chains. Then, two daughter chains could be formed on both parental templates, resulting in The formation of two DNA molecules identical to the original one" (J. Watson, 1968).

Fig. 20.3. Model of METABOLISM/36.html">DNA replication According to the Watson and Crick mechanism.

1, 2 — parental DNA strands; 1a, 2a — daughter DNA strands.

Semiconservative and conservative mechanisms of replication

The model of DNA duplication (replication) initially proposed by J. Watson and F. Crick was named the semiconservative mechanism of replication. According to this mechanism, the "parental" DNA molecule separates into two strands during replication, each of which serves as a template for the synthesis of the second strand based on THE PRINCIPLE OF complementarity, resulting in the formation of identical "daughter" molecules.

Along with the aforementioned Watson-Crick model, the conservative mechanism of replication was also considered as theoretically possible. According to this model, during Cell duplication, the "old" parental DNA molecule remains intact, while completely new DNA molecules are assembled from free mononucleotides:

Fig. 20.4. Theoretically possible mechanisms of DNA replication.

Experimental substantiation of the semiconservative mechanism of DNA replication was carried out in experiments by M. Meselson and F. Stahl (M. Meselson, F.W. Stahl; 1957). The main idea of M. Meselson and F. Stahl's experiment was based on the difference in specific density and, accordingly, the possibility of fractionating DNA molecules differing in the content of light (N¹⁴) or heavy (N¹⁵) nitrogen isotopes by density gradient ultracentrifugation in CsCl. The scheme of the experiment can be divided into the following stages:

Fig. 20.5. Scheme of the experiment by M. Meselson and F. Stahl.

1) cultivation of E. coli cells on a medium containing NH₄Cl with the light isotope (N¹⁴) as a nitrogen source; the density of "light" DNA molecules contained in such cells was determined (Fig. 20.5 a);

2) cultivation of E. coli cells for several generations on a medium containing NH₄Cl with the heavy isotope (N¹⁵) as a nitrogen source; the density of "heavy" DNA molecules contained in such cells was determined (Fig. 20.5 b);

3) E. coli cells containing "fully heavy" DNA molecules (N¹⁵-DNA) were transferred back to a medium with the light (N¹⁴) nitrogen isotope; after the first replication, daughter cells (F₁) were harvested, DNA was extracted from them, and its density was determined by ultracentrifugation. It was established that the density of this DNA from the F₁ generation was intermediate between the densities of "light" (N¹⁴) and "heavy" (N¹⁵) DNA molecules, meaning it consisted of half "light" and half "heavy" strands, which corresponded to the structure (N¹⁴, N¹⁵-DNA) (Fig. 20.5 c), thereby confirming the semiconservative mechanism of replication.