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

Chapter I. BIOMOLECULES AND CELLULAR STRUCTURES

CHAPTER 3. NUCLEIC ACIDS. NUCLEOTIDES

3.3. STRUCTURE, PROPERTIES, AND BIOLOGICAL FUNCTIONS OF DNA

Class="center">BIOLOGICAL Functions OF DNA:

1. Storage of Genetic information.

The amount of DNA in somatic and germ Cells of the human Organism is a constant value, which these cells receive during the Fertilization of parental Gametes and subsequent division of the zygote.

2. Transmission of genetic information to progeny.

The duplication of DNA molecules during Replication and the transmission of copies of parental molecules to progeny form The basis of the conservatism of heredity, preserving the fundamental biological characteristics of a species across many generations.

3. Expression of genetic information.

This biological function is carried out through The transfer of information encoded in DNA to messenger (template) RNA molecules (METABOLISM/31.html">Transcription) and the subsequent decoding of this information during Protein Synthesis (Translation).

The totality of these biological functions of DNA and the mechanisms of their Structure/175.html">Implementation has been termed the Central dogma of molecular biology (F. Crick) (Fig. 3.1):

Fig. 3.1. The central dogma of molecular biology.

Experimental proof of the genetic role of DNA (the transformation phenomenon)

In 1928, the English microbiologist F. Griffith, while studying two strains of the pneumococcus Streptococcus pneumoniae — a pathogenic strain causing Pneumonia in humans and mice (the encapsulated S-form) and a non-pathogenic mutant (the non-encapsulated R-form) — discovered The phenomenon of transformation. It consisted in the possibility of converting the non-pathogenic R-form into the pathogenic S-form:

Griffith established that pneumococcal transformation occurs under conditions of interaction within the organism of experimental animals (mice) between heat-killed S-form (pathogenic) and live non-pathogenic R-form cells. It was concluded that heat-killed virulent pneumococcal cells (strain S) contain a specific transforming factor which, upon penetrating live non-virulent cells (strain R), alters the biological Properties of the latter, conferring pathogenicity upon them, and this property is heritable:

Later, in 1944, a group of researchers from the Rockefeller Institute (USA) — O. Avery, C. MacLeod, and M. McCarthy — while investigating the Chemical Nature of extracts from pathogenic pneumococci that cause transformation, proved that the pneumococcal transforming factor is cellular DNA:

Thus, As a result of the research by O. Avery and co-workers, the genetic Concept of the "heredity factor" or "Gene" acquired a concrete molecular meaning for the first time — it turned out to be deoxyribonucleic acid.

Molecular weight and dimensions of DNA molecules

The molecular weight (m.w.) of Deoxyribonucleic Acids varies significantly across different biological objects: Viruses, prokaryotic, and Eukaryotic cells.

The precise Determination of the Molecular Weight of various DNA samples is hindered by the hydrodynamic fragility of giant nucleic acid molecules, especially in higher organisms, which break down into shorter fragments upon attempts to isolate them in an intact state. Furthermore, the DNA of many objects exhibits a complex molecular Organization, comprising a broad spectrum of various polynucleotide Conformations: linear single- and double-stranded molecules, circular single- and double-stranded molecules, and supercoiled structures.

However, the application of modern physicochemical Research Methods and Electron Cell/15.html">Microscopy has revealed that the molecular weight of DNA (calculated per single polynucleotide chain) typically ranges from 10⁶ to 10¹¹ daltons (Da).

DNA of Viruses and Prokaryotes

The smallest molecular weight and molecule length are found in the DNA of the simplest living entities—viruses, specifically bacterial viruses (Bacteriophages). For instance, the molecular weight of one of the smallest bacteriophages, phage ΦX 174, is reported by various studies to be 1.6 · 10⁶ (or 3.4 · 10⁶) Da.

In prokaryotic microorganism cells, the amount of DNA and its molecular organization are significantly higher than in viruses. Specifically, the DNA of the bacterium Escherichia coli (E. coli) is a covalently closed double-stranded circle with a molecular weight of 1.9 · 10⁹ (or, according to other sources, 26 · 10⁹) Da. As biological complexity increases in the transition from viruses to prokaryotes, so does the number of nucleotide pairs in double-stranded DNA molecules.

DNA of Eukaryotic Cells

Anucleate Prokaryotic Cells contain a single DNA molecule located in a specialized cytoplasmic region known as the nucleoid. In contrast, eukaryotic DNA is housed within The Nucleus and, outside of Cell Division phases, forms part of an amorphous nucleoprotein complex called nuclear Chromatin. During preparation for mitosis (in the S phase of the Cell Cycle), DNA replication takes place, followed by chromatin Condensation to form distinct cytological structures—Chromosomes—which concentrate The Cell's nuclear genetic material.

Through the replication of nuclear DNA during the S phase, each somatic cell acquires a diploid set of genetic material, ensuring its even distribution between two daughter cells. Consequently, during the metaphase stage of the cell cycle (the period immediately preceding the segregation of genetic material into daughter cells), each chromosome in a Introduction/5.html">Eukaryotic Cell contains two completely identical DNA molecules. The chromosome count in eukaryotic cells is species-specific. For example, cells of the classic genetic model organism, the fruit fly Drosophila melanogaster, contain 8 chromosomes, whereas human somatic cells have 46. The molecular weight of DNA from Human chromosomes averages 1.6 · 10¹¹ Da, which corresponds to 2.4 · 10⁹ Base Pairs. The physical length of fully extended DNA molecules from eukaryotic cells reaches several centimeters.

Molecular size parameters of DNA from various viral, prokaryotic, and eukaryotic representatives are summarized in Table 3.3.

Table 3.3. Molecular weight and dimensions of DNA molecules from various biological sources

As Table 3.3 demonstrates, overall, There is a direct proportionality between advancing evolutionary levels and biological complexity in living organisms and the amount of genetic material, expressed in terms of base pair count and, consequently, the molecular weight of DNA. The significantly smaller Molecular dimensions and nucleotide organization complexity of Mitochondrial DNA only reinforce the prevailing theory regarding THE ORIGIN OF these Organelles from primitive prokaryotes.

Secondary structure

Analysis of the Nucleotide Composition of DNA molecules from various biological sources has shown that, regardless of their origin (bacterial, plant, or animal), all DNA molecules exhibit specific quantitative relationships between their purine and pyrimidine nucleotide contents. According to these regularities (Chargaff's rules):

1) the sum of purine bases equals the sum of pyrimidine bases, i.e.:

A + G = T + C, or

2) the number of 6-amino groups equals the number of 6-keto groups (according to Fisher's chemical nomenclature);

3) the content of adenine equals that of thymine, and the content of guanine equals that of cytosine (the equivalence rule):

A = T, G = C.

These quantitative ratios among nitrogenous bases, combined with X-Ray Diffraction data on DNA Structure (M. Wilkins), enabled American biochemist James Watson and English physicist Francis Crick, working at the University of Cambridge, to propose a spatial double-helix model for the DNA molecule.

According to the Watson-Crick model, a DNA molecule consists of two chains forming a right-handed helix, with both polynucleotide chains coiled around a central axis; furthermore, the two polynucleotide chains in the DNA molecule are antiparallel (Figs. 3.2, 3.3).

Fig. 3.2. Schematic representation of a double-stranded DNA molecule.

Fig. 3.3. Antiparallel orientation of polynucleotide chains in a DNA molecule.

The Double Helix is stabilized by Hydrogen Bonds formed between oppositely positioned, so-called complementary nitrogenous bases (adenine paired with thymine, and guanine paired with cytosine, respectively), which accounts for the aforementioned empirical Chargaff's rules.

Besides hydrogen bonds, the Stability of the DNA molecule is also maintained by interactions between the $\pi$-electron clouds of the nitrogenous base heterocycles stacked vertically along the helix axis, known as "base stacking interactions".

Structural Features of the double helix: helix diameter is 20 μ; distance between adjacent nitrogenous bases along the helical axis is 3.4 Å; the helical structure repeats at intervals of 34 Å, which corresponds to every 10 nucleotide pairs.

These structural features refer to the B-form of the DNA molecule proposed by Watson and Crick (Fig. 3.4). However, depending on interactions with varying numbers of Water molecules and cations, DNA can adopt other structural forms—A, C, and Z—which may correspond to specific physiological conditions and the interaction of DNA with nuclear chromatin Proteins (C-form).

Fig. 3.4. B-form of the DNA molecule.

Tertiary structure

Within the living cell, the double helix representing the Secondary structure of DNA does not exist as an extended molecule, but is further folded in space to form specific tertiary structures known as supercoils.

In this supercoiled state, DNA molecules complexed with specific cellular proteins form the nucleoid in prokaryotes and nuclear chromatin in eukaryotes. Supercoiling enables long DNA molecules to form compact structures, such as nuclear chromosomes. For instance, as a result of compaction, a human nuclear DNA molecule approximately 8 cm long is fitted into a chromosome only 5 nm in length.

Physicochemical properties

Reactivity

All polynucleotides, and DNA in particular, are strong polybasic acids with a low pK value. The acidity of DNA is due to secondary phosphate groups that are fully ionized at pH > 4.

Due to their acidic properties and the presence of negative charges on their surface, DNA molecules at physiological pH actively react and form complexes with cations:

- Polyamines (spermidine, spermine);

- basic proteins (Histones, protamines);

- metal cations (Ca²⁺, Mg²⁺, Fe²⁺).

Fig. 3.5. Schematic representation of the interaction between a DNA polynucleotide chain and basic proteins.

Viscosity and optical activity

The high molecular weight and great length of DNA molecules account for the high viscosity of even their very dilute solutions. The viscosity of DNA molecules in solution depends on their conformation and changes significantly under conditions of Denaturation and renaturation (see below), which allows viscometric methods to be used for studying The kinetics of these processes.

Owing to their ordered secondary structure, DNA molecules are optically active, meaning they are capable of rotating the plane of polarized light. The optical activity of DNA solutions is also utilized to monitor Conformational Changes in the molecules.

UV absorption

Nitrogenous compounds (and their corresponding NUCLEOTIDES) that make up DNA and RNA Nucleic Acids have the property of absorbing ultraviolet light at 260 nm.

When polynucleotides are formed, the mutual influence of parallel-aligned base pairs along the DNA molecule is accompanied by a noticeable decrease in UV absorption. Thus, the absorbance at 260 nm of native DNA is somewhat lower (on average by 40%) than the absorbance of the sum of the nitrogenous bases comprising the polynucleotide—the hypochromic effect. Conversely, disruption of the highly ordered double-helical conformation of DNA and the structural relationships between the nitrogenous bases leads to a hyperchromic effect, i.e., an increase in the absorbance of DNA solutions at 260 nm, which makes it possible to study The process of denaturation.

Denaturation

DNA denaturation refers to the disruption of the native double-helical conformation of DNA molecules and their ordered spatial arrangement, resulting in The formation of random single-stranded coils. Under denaturing conditions, the covalent bonds within DNA remain intact, but the double helix unwinds, leading to the loss of specific interactions between nitrogenous bases. Renaturation is the restoration of the native secondary conformation of DNA, which occurs under certain specific conditions.

DNA denaturation is accompanied by a hyperchromic effect and a decrease in the viscosity of its solutions (Fig. 3.6).

Fig. 3.6. Hyperchromic effect during the denaturation of microbial DNA.

Nucleic acids that have undergone denaturation lose their biological properties.

The Molecular Basis of DNA denaturation is the disruption of hydrogen bonds between the complementary nitrogenous bases A-T and G-C, respectively.

Structural Changes in DNA molecules that lead to their denaturation occur as a result of:

- sharp changes in pH toward acidic or alkaline values;

- heating DNA solutions to specific temperatures.

Thermal denaturation of DNA is commonly referred to as melting. Each type of DNA molecule (i.e., molecules with a specific nucleotide composition) is characterized by a specific denaturation Temperature, known as the melting temperature (or melting point) — T_m. The T_m of different DNA samples depends on The ratio of G-C and A-T base pairs in their composition.

The ratio between G-C and A-T pairs is an important indicator of the nucleotide composition of DNA molecules from various biological sources. Since three Hydrogen bonds are formed between guanine and cytosine in a double-stranded DNA molecule (as opposed to two between adenine and thymine), the thermal dissociation of a G-C pair requires more energy, meaning it occurs at higher temperatures than the disruption of an A-T pair. Consequently, the melting temperature of DNA molecules is directly proportional to their G-C content, which makes determining T_m a useful method for estimating DNA nucleotide composition.