DNA molecules in eukaryotic cells are very large. Thus, the length of DNA molecules isolated from human cells reaches several centimeters. It is generally accepted that each eukaryotic chromosome contains one - the only continuous DNA molecule. Considering the species number of chromosomes in mammals, it can be said that, on average, they have about 2 m of DNA per interphase nucleus, which is located in a spherical nucleus with a diameter of less than 10 μm. At the same time, a certain order of arrangement of DNA molecules must be preserved in the nucleus in order to ensure its ordered functioning.

DNA molecules in the nuclei of eukaryotic cells are always in complex with proteins in the composition of chromatin, which is formed from chromosomes after the end of nuclear division as a result of complex process unwinding (despiralization) of chromosomes.

Proteins account for about 60% of the dry weight of chromatin. The proteins in its composition are very diverse. They are usually divided into two groups: histones and non-histone proteins. It is histones, characteristic only for eukaryotic cells, that carry out the first stages of DNA packaging, which are very similar in most of the studied objects.

Histones account for up to 80% of all chromatin proteins. Their interaction with DNA occurs due to ionic bonds and does not depend on the sequence of nucleotides in the DNA molecule. Histones are not very diverse. These are globular proteins represented by 5-7 types of molecules. The best known classes of histones are HI, H2A, H2B, H3, and H4. Them basic properties determined relative to high content basic amino acids: lysine and arginine (Fig. 3.7). Positive charges on the amino groups of these amino acids provide an electrostatic bond between histones and negative charges on the phosphate groups of DNA. Of all the nuclear proteins, histones are the best studied. Their molecular weight is relatively small (the maximum is for the H3 histone - 153 thousand daltons). In almost all eukaryotes, they have similar properties and are divided into the same classes. Of the studied proteins, these proteins are the most conservative: their amino acid sequences are similar even in distant species. The exception is HI histones, which are characterized by significant interspecies and intertissue variations.

During the life of cells, histones can undergo post-translational modifications, which change their properties and ability to bind to DNA. Histones are synthesized in the cytoplasm, transported to the nucleus, and bind to DNA during its replication in the S-period of the cell cycle. Histones incorporated into chromatin are very stable and have low speed exchange.

The presence of histones in all eukaryotic cells, their similarity even in very distant species, their mandatory composition of chromosomes and chromatin - all this indicates the extremely important role of these proteins in the life of cells. A landmark event in the study of DNA packaging in chromatin was the discovery of particle nucleosomes, in which the first stage of DNA packaging in chromatin occurs. The core of the nucleosome is always conservative, contains eight molecules: two molecules of histones H4, H3, H2A, H2B each. On the surface of the core is a DNA segment of 146 nucleotide pairs, forming 1.75 turns around the core. A small section of DNA remains unbound to the core, it is called a linker (Fig. 3.8). In different objects, the linker site can vary from 8 to 114 nucleotide pairs per nucleosome.

It is calculated that the entire haploid human genome (3 x 109 base pairs) accounts for 1.5 x 107 nucleosomes. General form chromatin, represented by a DNA molecule packaged using nucleosomal structures, can be compared to beads on a string (Fig. 3.9). Nucleosomes are capable of self-assembly in the presence of DNA and histones in a test tube in a certain ratio. The first nucleosomal level of DNA compaction increases the packing density of DNA by 6-7 times.

The nucleosomal structure of chromatin is involved in the next stage of packaging with the help of histone HI, which binds to the linker portion of DNA and the surface of the nucleosome. Due to the complex interaction of all components, an ordered structure of a spiral type arises, which is often called a solenoid (Fig. 3.10). It increases the compactness of DNA by another 40 times. Because the solenoid structure has a reduced ability to bind to proteins that mediate transcription, it is believed that this level of DNA compaction may act as a gene-inactivating factor. Some authors consider the solenoid structure as one of the options chromatin packaging

with the help of histone HI and consider the existence of other morphological variants, for example, the nucleomer, or superbeads, to be probable (Fig. 3.11).

More high levels DNA compaction in chromatin is associated with non-histone proteins. They account for about 20% of all chromatin proteins. This assembled group of proteins is distinguished by a wide range of properties and functions. In total, the fraction of non-histone proteins combines about 450 individual proteins, the properties and specific functions of which have not yet been sufficiently studied. It was found that some of them specifically bind to certain DNA regions, as a result of which chromatin fibrils form loops at the sites of DNA binding to non-histone proteins. Thus, higher levels of DNA packaging in chromatin are provided not by spiralization of chromatin threads, but by the formation of a transverse looped structure along the chromosome (Fig. 3.12). At all these stages of DNA compaction, chromatin is present in an active form; transcription and synthesis of all types of RNA molecules take place in it. Such chromatin is called euchromatin. Further packaging of chromatin leads to its transition to an inactive state with the formation of heterochromatin.

This process is associated with the spiralization of loop groups and the formation of rosette-like structures from chromatin fibrils, which have optical and electron density and are called chromomeres (Fig. 3.12). It is assumed that along the chromosome is located a large number of chromomeres interconnected into a single structure by sections of chromatin with pucleosomal or solenoidal DNA packaging. Each pair of homologous chromosomes has its own chromomeric pattern, which can be detected using special staining methods, provided that the chromatin is siralized and it passes into the state of chromosomes.

The loop-rosette structure of chromatin provides not only packaging of DNA, but also organizes functional chromosomes, since at their bases DNA loops are associated with non-histone proteins, which may include replication enzymes that provide DNA duplication, and transcription enzymes, due to which all types of synthesis occur. RNA.

DNA regions packaged in the form of heterochromatin can have a dual nature. There are two types of heterochromatin: facultative and constitutive (structural). Facultative heterochromatin is a part of the genome that is temporarily inactivated in certain cells. An example of such chromatin is the sex heterochromatin of the inactivated X chromosome in somatic cells of women. Structural heterochromatin in all cells is constantly in an inactive state and probably performs structural or regulatory functions.

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Chromosomal theory of heredity
Lesson 6. Inheritance of sex-linked traits ………………………………………. Lesson 7. Peculiarities of inheritance of genes localized in one chromosome …………… Lesson 8. Kartirova

Population genetics
Occupation. Genetic structure of the population (cross-breeders and self-pollinators) 1. Define a population. Describe populations according to the type of reproduction of organisms.

Material foundations of heredity
The concept of genetic information. Evidence of the role of the nucleus and chromosomes in the phenomena of heredity. Localization of genes in chromosomes. The role of cytoplasmic factors in the transmission of hereditary information

Genetic analysis
Basic patterns of inheritance. Goals and principles of genetic analysis. Methods: hybridological, mutational, cytogenetic, population, twin, biochemical, statistical. G

Extra-nuclear inheritance
Patterns of non-chromosomal inheritance, unlike chromosomal inheritance. Research methods: reciprocal, reciprocal and absorbing crosses, transplantation method, biochemical methods.

genetic variability
The concept of hereditary and non-hereditary (modification) variability. Formation of traits as a result of the interaction of the genotype and environmental factors. The reaction rate of the genotype. Adaptive character

Fundamentals of Molecular Genetics
Representation of the Morgan school on the structure and function of the gene. Study of the fine structure of the gene on the example of the T4 phage (Benzer). Gene as a unit of function (cistron). Overlapping of genes in one section of DNA. Int

population genetics
The concept of species and population. Population as a natural-historical structure. The concept of the frequencies of genes and genotypes. Mathematical models in population genetics. Hardy-Weinberg law, possibly

human genetics
Features of a person as an object of genetic research. Methods for studying human genetics: genealogical, twin, cytogenetic, biochemical, ontogenetic, population. use

History of human genetics
The successes of human genetics, its history, are closely connected with the development of all sections of genetics. Long before the discovery of G. Mendel, various authors described pathological hereditary traits in h

genealogical method
The basic patterns of heredity established for living organisms are universal and fully valid for humans. However, as an object of genetic research, a person has

twin method
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Population-statistical method
One of important directions in modern genetics is population genetics. It studies the genetic structure of populations, their gene pool, the interaction of factors that determine the constancy

Cytogenetic method
The basis of the method is the microscopic study of human chromosomes. Cytogenetic studies have been widely used since the early 1920s. 20th century for studying the morphology of human chromosomes, counting xp

Method of genetics of somatic cells
The fact that somatic cells carry the entire amount of genetic information makes it possible to study the genetic patterns of the whole organism on them.

Biochemical method
The cause of many congenital metabolic disorders are various enzyme defects resulting from mutations that change their structure. Biochemical parameters (primary gene product, on

Molecular genetic methods
The end result of molecular genetic methods is the detection of changes in certain sections of DNA, gene or chromosome. They are based on modern techniques work with DNA or RNA. In the 70-80s. in St.

Chemical composition and structure of the DNA molecule
The founder of genetics Gregor Mendel in 1865 proved for the first time that each trait of an organism is determined by a pair of hereditary factors. At the beginning of the XX century. paired hereditary factors received on

Organization of genetic material in human chromosomes
The general organization of human chromosomes is traditional: in metaphase, the chromosome consists of two sister chromatids connected to each other in the region of the primary constriction (centromere). The centromere divides chro

human chromosomes
The history of the development of human cytogenetics For the first time human mitotic chromosomes were described in the works of J. Arnold (1879) and W. Flemming (1882). In subsequent years, various estimates of their number

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At the turn of the 70s. 20th century molecular genetics has reached a certain completion in its development: the structure and mechanism of DNA replication have been established, the "central dogma" has been proclaimed

The study of human genomes
The last decades at the turn of two epochs are reflected in the rapid growth in the field of higher human biology. This is due, initially, to the work on deciphering the human genome, carried out in the past.

Introduction

DNA molecules in eukaryotic cells are very large. Thus, the length of DNA molecules isolated from human cells reaches several centimeters. It is generally accepted that each eukaryotic chromosome contains one single continuous DNA molecule. Taking into account the species number of chromosomes in mammals, we can say that, on average, they have about 2 m of DNA per interphase nucleus, which is located in a spherical nucleus with a diameter of less than 10 μm. At the same time, a certain order of arrangement of DNA molecules must be preserved in the nucleus in order to ensure its ordered functioning.

That is why DNA molecules in the nuclei of eukaryotic cells are always in complex with proteins in the composition of chromatin, which is formed from chromosomes after the end of nuclear division as a result of a complex process of unwinding (despiralization) of chromosomes.

Exploring structural organization chromatin and chromosomes, one can definitely speak of several levels of DNA compaction. The first one is nucleosomal, giving a seven-fold compaction of DNA and the composition of DNP fibrils, the second is a fibril with a diameter of 30 nm, or the nucleomeric level, with a 40-70-fold degree of packing, the third is domain-loop, or chromomeric, leading to 600-700 -fold compaction of DNA within these structures. To maintain the first two levels of compactization, the participation of only histone proteins was sufficient, while loop and rosette-like domain structures already required the participation of non-histone proteins and the transition from the helical or solenoidal type of DNA folding to the formation of compact globular structures consisting of loops of chromatin fibrils with a diameter of 30 nm , to structures of the chromomeric type, already having dimensions of 0.1-0.2 μm.

DNA packaging in chromosomes

Compactness is the fundamental difference between the eukaryotic genome and the prokaryotic genome. With an average difference in genome sizes of 3 orders of magnitude, the linear sizes of eukaryotic chromosomes are commensurate with the length of prokaryotic DNA.

There are at least 4 levels of DNA compaction. In this case, the DNA strand is "shortened" by 10,000 times. It is similar to putting a thread as long as the Ostankino Tower (500 m) into a matchbox (5 cm).

The chromosomes of eukaryotic cells consist mainly of chromatin, a complex of double-stranded DNA, and five histone proteins, designated H1, H2A, H2B, H3, and H4.

It is histones that provide the first two levels of compaction of the eukaryotic genome - nucleosomal and nucleomeric.

General characteristics of histones

Histones are the main proteins. All of them are enriched with lysine and arginine - positively charged amino acids. Depending on the ratio of amino acids in the structure of histones, 5 histone fractions are usually isolated. A lot of them are produced - 60 million molecules of each fraction per cell.

Histone modifications have a very strong effect on DNA compaction. Histones can be methylated, phosphorylated (by serine, threonine, tyrosine), i.e. amino acid residues are easily modified. In addition, alkylation and acetylation of histones is possible.

Major histone fractions:

All histones, except for H1, are extremely conserved in evolutionary terms (in cow and clover, the difference in H2A is only one amino acid!). Consequently, these proteins perform a fundamental function that is provided in the same way in all eukaryotes. Any mutation in the histone genes is lethal.

H1 is a very variable fraction. This histone is different not only in species, but even in one organism, depending on the stages of ontogeny.

In histones, lysine and arginine are clustered. The middle part of the histone contains hydrophobic amino acids. Positively charged histone amino acids provide electrostatic interactions with DNA. The central part is necessary for the interaction of histones with each other.

The role of histones in DNA folding is important for the following reasons:

1) If chromosomes were only stretched DNA, it's hard to imagine how they could replicate and separate into daughter cells without getting tangled or broken.

2) In an extended state, the DNA double helix of each human chromosome would cross the cell nucleus thousands of times; thus, histones package a very long DNA molecule in an orderly manner into a nucleus several micrometers in diameter;

3) Not all DNA is folded in the same way, and the nature of the packaging of a region of the genome into chromatin probably affects the activity of the genes contained in this region.

1) Nucleosomal - at this level, the DNA double helix is ​​wound around a protein complex containing 8 histone molecules - proteins with an increased content of positively charged amino acid residues of lysine and arginine. These are the histones H2B, H2A, H4 and H3. A structure with a diameter of 11 nm is formed, resembling beads on a string. Each "bead" - the nucleosome contains about 150 base pairs. The nucleosomal level gives a shortening of the DNA molecule by 7 times. During replication, this level of packaging is removed, while during transcription, nucleosomes are preserved.

2) At the second level, nucleosomes approach each other with the help of histone H1, resulting in the formation of a fibril with a diameter of 30 nm. The reduction in the linear size of DNA occurs by 6-10 times. This level of packaging, like the first one, does not depend on the primary structure of DNA.

3) Loop level. Provided by non-histone proteins. They recognize certain DNA sequences and bind to them and to each other, forming loops of 20-80 thousand bp. Shortening due to loops takes place 20-30 times. A typical mammalian chromosome can contain up to 250 loops.

4) metaphase chromosome. Before cell division, DNA molecules are doubled, the loops are stacked, the chromosome thickens and is visible under a light microscope. At this packing level, each chromosome consists of two chromatids. Each chromatid contains one DNA molecule.

Functions of DNA.

1. DNA is the carrier of genetic information. The function is provided by the fact of existence genetic code.

2. Reproduction and transmission of genetic information in generations of cells and organisms. The function is provided by the process replication .

3. Implementation of genetic information in the form of proteins, as well as any other compounds formed with the help of enzyme proteins. Function provided by processes transcriptions and broadcasts .

Directly from the structure of DNA follows the mechanism of its exact reproduction (replication). DNA structure replication is based on the principle complementarity : in a double helix, two DNA polymer chains are connected to each other due to the formation of pairs G - C, C - G, A - T, T - A. If two chains of the double helix diverge, then a new complementary chain can be built on each of them - opposite G of the original chain, the C of the new chain will be established, opposite the C of the old chain - G of the new chain, opposite A - T, and opposite T - A. As a result, two child double helixes will be obtained, completely identical to the original - parent.

Ribonucleic acids ubiquitous in nature. biological function RNA is due to the fact that they ensure the implementation in the cell of hereditary information that is transmitted using DNA.

There are three main types of RNA in the cell: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

RNA is a polynucleotide similar to DNA, but with its own characteristics.

1) The carbohydrate in RNA is represented by ribose, which has a hydroxyl group in the second position of the carbon atom.

2) Unlike DNA, the molecules of all three types of RNA are single-stranded, which is one of the important features of RNA. Besides, distinctive feature RNA is that it is not characterized by a stable helical structure.

3) RNA contains 4 nitrogenous bases - adenine, cytosine, guanine and uracil.

The following general principles of the structure of all types of RNA are distinguished:

1) RNA is a single-stranded polynucleotide.

2) RNA forms a secondary structure - a set of short helical sections, which are formed due to antiparallel complementary pairing of adjacent segments of the chain.

3) RNA is able to form a tertiary structure due to long-range complementary interactions within the strand and interstrand interactions.

4) High polymeric RNA is able to fold into compact particles.

5) RNA has significant conformational mobility.

© Razin S.V.

Spatial organization of DNA

S.V. Razin

Sergei Vladimirovich Razin, Corresponding Member of the Russian Academy of Sciences, Doctor of Biological Sciences,
Head of the Laboratory of Structural and Functional Organization of Chromosomes at the Institute of Gene Biology, Russian Academy of Sciences,
Professor of the Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University.

Even at the beginning of the last century, thanks to the use of purely genetic methods, it became clear that genes are linearly located on chromosomes. Since then, most researchers have considered the genome as a chain of successively located genes and intergenic regions, including various regulatory and other (seemingly insignificant) sequences. Such a stereotype of thinking is reflected, in particular, in the fact that the distances between genes or other sections of DNA are usually indicated in thousands of nucleotide pairs, meaning the distances along the DNA molecule.

Although this is quite correct, such an idea of ​​the linearity of the genome contains certain dangers. The fact is that in the nucleus of a eukaryotic cell, the genome is packed extremely complex. As a result, DNA sequences, including genes, separated from each other by tens or hundreds of thousands of nucleotide pairs, and sometimes even located on different chromosomes, are in close proximity in three-dimensional space. This ensures the interaction of protein complexes associated with remote (if we count along the DNA molecule) regulatory elements. Such interactions significantly expand the possibilities of operation of various regulatory systems in the eukaryotic cell genome. AT last years Several fundamentally new observations have been obtained that have significantly increased interest in the spatial organization of DNA in the nucleus. We will try to summarize modern achievements in this area.

DNA packaging in the nucleus

In an average eukaryotic cell, the total length of genomic DNA is about 2 m, and the diameter of its nucleus is only ~10–20 µm. In this case, the totality of genes operating in a given cell must be accessible to RNA polymerases and transcription factors, and all DNA in dividing cells must be replicated.

Today it is known that DNA packaging in the nucleus of a eukaryotic cell is carried out in several stages (Fig. 1). First, the DNA strand fits into nucleosomes, while its length decreases by six to seven times. Then the nucleosomal thread is folded into the so-called 30 nm fibril (solenoid or zigzag thread), which provides additional compaction by 40 times. Further, the fibril is organized into large (50 or more thousand base pairs) loops, the ends of which are fixed on the protein skeleton of the nucleus (it is often called the nuclear matrix). At this stage, the linear dimensions of DNA are reduced by 700 times. There are also the following levels of DNA compaction, information about which is currently very scarce and contradictory.

Rice. one. DNA packaging levels in the nucleus of eukaryotic cells.

So far, we have been talking only about the packaging of one extended DNA molecule. In the first approximation, this can be considered the DNA of one chromosome. However, the genome of a eukaryotic cell is divided into several chromosomes. For example, in the cells of a favorite object of geneticists - fruit fly Drosophila - there are four pairs of chromosomes (there are 46 in human cells). Individual chromosomes can only be seen under a microscope during mitosis. At other phases of the cell cycle, they are not visible, and the cell nucleus appears to be relatively homogeneous. For many years, molecular biologists have been interested in the question of whether individual chromosomes occupy limited spaces within the nucleus, or if chromosomes are decompacted, the DNA of each of them is distributed throughout the nucleus, inevitably mixing with the DNA of other chromosomes.

Rice. 2. Staining of chromosome territories.

A - DNA of human metaphase chromosomes. B - DNA of the interphase nucleus. B - DNA of metaphase chromosomes ( Blue colour) after hybridization with chromosome-specific probes recognizing chromosome 18 (red) and chromosome 19 ( green color); two homologues of the corresponding chromosome are shown. D - results of DNA hybridization of interphase nuclei with chromosome-specific probes recognizing chromosomes 18 and 19. Eight sections of the nucleus were made using a confocal microscope; all nuclear DNA is stained blue (as in Fig. B).

About 10 years ago, the answer to this question was found. Molecular hybridization methods made it possible to stain individual chromosomes in the interphase nucleus (Fig. 2). It turned out that, contrary to the generally accepted point of view at that time, they occupy limited non-overlapping spaces inside the nucleus (called "chromosomal territories", Fig. 3) and are located in a non-random way: chromosomes rich in genes are localized closer to the center of the nucleus, and poor in genes - closer to its periphery. The nuclear matrix plays an important role in maintaining specific positions of chromosome territories.

Rice. 3. 2D and 3D models of the nucleus showing the location of chromosome territories.

Interaction of remote regulatory elements

The packaging of DNA into hierarchical chromatin structures is fundamentally important for the physical distance between regulatory sequences and their orientation in space. And these sequences always serve as binding sites for regulatory proteins. Even the organization of DNA into nucleosomes can make these sites inaccessible to protein factors, or orient them in such a way that the protein complexes planted on them, due to purely steric reasons (for example, orientation to opposite sides) will not be able to interact with each other. And with the formation of fibrils, the possibilities of suppressing or activating certain regulatory systems increase. However, the arrangement of nucleosomes on DNA is quite dynamic. Reversible changes in their structure and the degree of chromatin condensation (in particular, the transition from an unfolded nucleosomal filament to a 30 nm fibril and more compact heterochromatic structures) constitute the most studied part of epigenetic mechanisms.

We will not discuss these mechanisms, but dwell on the next level of DNA packaging in chromatin, namely, on extended DNA loops (Fig. 1). They can be seen by electron microscopy of metaphase chromosomes and interphase nuclei from which histones have been removed. The presence of topologically closed DNA loops in interphase nuclei has also been demonstrated using biochemical methods.

DNA loops attached to the nuclear matrix have interested specialists primarily because they could correspond in size to the functional units of the genome. To test this assumption, it was necessary to study the specificity of the organization of DNA into loops. First, to establish whether the division of the genome into loops is the same in all cells. If this assumption is correct, then some fragments of the genome should always be located at the bases of the DNA loops, while others should be in the loops themselves. Secondly, to find out if there are some special DNA sequences responsible for "anchoring" the loops on the protein matrix of the nucleus.

Genome Slicing Method

Over the past 30 years, several methods have been proposed for mapping the sites of attachment of DNA loops to the nuclear matrix (chromosomal backbone). Although these methods differ in details, they can be divided into two fundamentally different groups. The first is based on the isolation of the so-called "nuclear matrix adjacent DNA" (i.e., located at the bases of the loops) and the fraction of DNA loops that is cleaved from the nuclear matrix during limited treatment of the nuclei with the nuclease enzyme (Fig. 4). The preferential presence of the studied DNA fragment in the fraction adjacent to the nuclear matrix, obtained after a sufficiently intensive nuclease treatment, suggests that it is attached to the nuclear matrix. The second group of methods is aimed at studying the specificity of DNA sequences interacting with the nuclear matrix. All methods are based on selective binding. in vitro(i.e. in vitro) fragments of cloned DNA with an isolated nuclear matrix. However, the results obtained using the two methodological approaches turned out to be rather contradictory.

Rice. four. Scheme for determining the positions of genes in DNA loops.

A - nucleoid obtained after extraction of histones from nuclei not treated with nuclease; one of the DNA loops contains three genes: "a" is in the proximal (with respect to the nuclear matrix) part of the loop, "b" is in an intermediate position, and "c" is in the distal part;

B - after the treatment of nuclei with nuclease, approximately two breaks per loop are formed; DNA fragments located at the bases of the loops (inside the dotted circle) are attached to the nuclear matrix. After differential centrifugation, the fragments are separated, and the genes "a" and "b" remain in the DNA attached to the nuclear matrix;

B - after additional treatment with nuclease, only gene "a" remains.

We noticed that all methods are aimed at identifying and characterizing DNA fragments localized at the bases of the loops. Our fundamentally new approach is based on cutting the entire genome into loops and their subsequent characterization. At first glance, dividing the genome into individual loops is extremely difficult. It is very important here which tool to make breaks in the bases of DNA loops. Fortunately, nature itself provided us with such a tool. Studies have shown that one of the main components of the nuclear matrix is ​​the DNA topoisomerase II enzyme, which regulates the topology of DNA. This enzyme introduces double-strand breaks in DNA, which, after removal of topological stresses or separation of catenanes, are sutured (ligated) by the same enzyme. Throughout the reaction, the two-subunit enzyme remains bound to the DNA.

There are a number of inhibitors of DNA topoisomerase II (in our case, VM-26), which stop the reaction at the stage of the intermediate enzyme-DNA complex. (Interestingly, most of them are used as antitumor agents.) In this case, each of the enzyme subunits remains covalently bound to the 5' end of the broken DNA strand. If such blocked complexes are treated with a denaturing agent and then the enzyme is destroyed, then a DNA preparation will be obtained, cut into fragments at the sites of DNA contact with the enzyme (Fig. 5). If topoisomerase II were present only in the nuclear matrix, then a simple treatment of living cells with its inhibitors would cut the entire genome along the sites of DNA attachment to the nuclear matrix. However, the task is complicated by the fact that this enzyme is present in the nucleoplasm in a soluble form and can introduce breaks anywhere (if it appears next to the DNA at the moment the cells are treated with an inhibitor). The most likely breakpoints will be nucleosome-free regions most sensitive to DNA nuclease (DNase I). To rule out the possibility of breaks outside the DNA attachment sites of interest to the nuclear matrix, we extracted the soluble enzyme, and at the same time histones, by treating the nuclei with 2M NaCl (Fig. 4). The resulting so-called nucleoids were treated with DNA topoisomerase II inhibitors. So we managed to cut the entire genome into separate loops and their oligomers.

Rice. 5. Diagram of DNA loop mapping method:

A - the reaction catalyzed by DNA topoisomerase II and the mechanism of DNA cutting upon inhibition of the cross-linking activity of the enzyme VM-26 and other "topoisomerase poisons";

B - cutting of genomic DNA into individual loops. After removal of histones, unfolded DNA loops are still attached to the nuclear matrix (yellow circles) containing DNA topoisomerase II (purple circles). Nucleoids are incubated in VM-26 medium and then lysed with sodium dodecyl sulfate (SDS). At the sites of loop attachment to the nuclear matrix, topoisomerase II cuts DNA;

C - breaks appear in DNA loops treated with Sfi I restriction enzyme. The Sfi I-Sfi I fragment (shown in blue) can be identified by hybridization with a probe complementary to one of the ends of the full length restriction fragment (blue arrow). On the right is the same region of the genome after an additional break caused by topoisomerase II (purple circle). The size of the truncated fragment is equal to the distance from the site of cleavage by DNA restrictase Sfi I to the site of cleavage by its topoisomerase II;

D - a typical picture of the result of electrophoresis. All lanes show a full-length Sfi I-Sfi I DNA fragment. In the lanes containing DNA from nucleoids treated with high concentrations of VM-26, an additional (Sfi I-Topo II) fragment appears, which indicates that inside the studied DNA fragment there is an attachment site to the nuclear matrix.

What to do next? How to set the positions of the ends of the loops on the physical map of the genome? Recall that such a map shows the real distances along the DNA molecule between certain markers. Physical maps of various genomes began to be created long before the decoding of the genomes of humans and a number of other organisms. DNA cleavage sites by restriction enzymes are usually used as markers when creating such maps. Set the position of the site of attachment of the DNA loop to the nuclear matrix on physical map- means to determine the distance from the site of attachment to the site of DNA cleavage by one or another restriction enzyme. To do this, you can use the method of indirect labeling of the ends of DNA fragments, proposed about 30 years ago to map the positions of sites of hypersensitivity to DNase I.

The principle of this method lies in the fact that after introducing breaks into the DNA by one or another agent (in our case, by DNA topoisomerase II of the nuclear matrix), the preparation is additionally cut with the selected restriction enzyme. After separating the fragments by electrophoresis and transferring them to a nitrocellulose filter, hybridization is carried out with a sample complementary to the end of the cut fragment, inside which there may be an additional gap. If there is no such gap, then after hybridization a full-sized fragment will be obtained. But if inside this DNA fragment was cut by nuclear matrix topoisomerase II or another enzyme, the fragment will be shorter, and its length is equal to the distance from the site of DNA cleavage by the restriction enzyme to the site of DNA cleavage by the studied agent (Fig. 5). When working with loops excised by DNA topoisomerase II, the main difficulty is the need to separate very long DNA fragments according to size. This problem can be solved using pulsed field electrophoresis, which allows the separation of DNA fragments ranging in size from several thousand to several million nucleotide pairs.

Map of organization into loop-domains of the human dystrophin gene

We have successfully used the above method to map loop boundaries in a number of regions of the human and Drosophila genomes. After that, a large-scale task was set - to build a map of the organization into loop-domains of the longest known gene - the human dystrophin gene. This gene, located on the X chromosome, has about 2500 thousand base pairs, and its mRNA size is only 14 thousand base pairs. In other words, more than 99% of the total length of the gene is occupied by non-coding sequences (introns). Various rearrangements often occur in the dystrophin gene, some of which lead to severe hereditary diseases - muscular dystrophies.

Rice. 6. Map of the loop organization of the human dystrophin gene.

Up- diagram of the location of sites and regions of DNA attachment to the nuclear matrix (horizontal lines marked with numbers 1-8) within the boundaries of the dystrophin gene. Vertical arrows (Latin letters A-I) indicate the location of cleavage sites; horizontal - at the position of hybridization samples.

At the bottom- visualization scheme of unique DNA fragments on nucleoid preparations. After extraction from the histone nuclei, the DNA loops unfold and form a crown around the nuclear matrix (a). The preparations are hybridized with samples containing biotin (bold line in scheme b). Such a sample can be seen after staining with antibodies bound to a fluorescent dye (black circles in scheme c).

A map of the dystrophin gene cleavage by SfiI was constructed even before the determination of the complete nucleotide sequence of the human genome. We mapped the positions of DNA attachment sites to the nuclear matrix relative to DNA cleavage sites by this restriction enzyme and found that the dystrophin gene has at least nine loops separated by eight attachment zones. In some cases, the length of DNA segments separating two adjacent loops is comparable to the length of the loops themselves (Fig. 6a). This fundamentally new observation made it possible to consider the zones of DNA attachment to the nuclear matrix as a special part of the genome. Curiously, this is where the recombination hotspots previously identified in the dystrophin gene are located. The discovered pattern turned out to be true for a number of other studied genes. Another interesting observation (also confirmed in other experimental models) is that the sites of loop attachment are the sites from which DNA replication begins. This confirms the position we formulated 20 years ago about the most important principle of organization of the eukaryotic chromosome - its construction from structural and functional domains corresponding to replication units.

Loops of DNA under a microscope

The experimental approach that we used to map DNA loops is based on a number of logical assumptions arising from the radial-loop model of the chromosome structure. Until recently, there was no direct evidence that DNA loops mapped with different methods, exactly those that can be seen on cytological preparations. Among the many intertwining loops of DNA observed under electron microscope, it is almost impossible to identify the loop as a genome fragment of interest to the researcher. However, this is possible when analyzing loops with lower resolution.

Rice. 7. Photomicrographs of hybridization results in situ with preparations of nuclear halo (a) with a fragment of the human genome - a DNA loop mapped in the dystrophin gene. This loop is limited by nuclear matrix attachment regions 7 and 8. Hybridization without competitive DNA (b) and in the presence of an excess of unlabeled fraction of repetitive human DNA sequences (c).

If you look at the nuclei extracted with 2M NaCl in a fluorescent microscope (after staining DNA with one or another fluorescent dye), you can see the crown of DNA loops in the form of a cloud surrounding a more brightly colored central zone (nuclear matrix) (Fig. 7, a and diagrams on Fig.6). Such drugs are called nuclear halos (nuclear halos), on which individual loops are indistinguishable. To see them, you need to use the hybridization method. in situ(in this case preparations of nuclear halos immobilized on glass) with a genome fragment of interest. The sample should contain nucleotide analogs (eg biotinylated uridine) that stain with fluorescent dyes such as red or green after hybridization. This allows you to simultaneously analyze the distribution of DNA, which is most easily stained with DAPI (4',6-diamidino-2-phenylindole) in lilac, and the distribution of the sample after hybridization, stained in red or green.

During the implementation of the human genome sequencing program, thousands of extended (100-300 thousand base pairs) fragments of human DNA were cloned in different laboratories. Most of the clones were systematized according to the positions of the cloned DNA fragments in the human genome. There is a range scientific centers, where you can buy a clone of interest. We took a cloned fragment of human DNA representing the DNA loop mapped by us in the dystrophin gene, limited by attachment sites 7 and 8 (see Fig. 6). After hybridization of this fragment with preparations of nuclear halo, many signals are revealed, distributed over the entire field (Fig. 7b). This is due to the fact that in the DNA of higher eukaryotes, including humans, there are many repetitive sequences distributed throughout the genome.

The repeats in our sample hybridize with all complementary sequences. It is clear that the results of such an experiment cannot be interpreted. Fortunately, signals from hybridization of repeated sequences can be suppressed. To do this, we carried out hybridization in the presence of an excess of an unlabeled fraction of repetitive human DNA sequences and saw DNA loops attached to the nuclear matrix (Fig. 7c). All of them had the same size (within the measurement error), corresponding to the length of the DNA fragment mapped in experiments on excision of loops by DNA topoisomerase II of the nuclear matrix.

The significance of this result goes beyond a simple confirmation of the correctness of our map of the domain organization of the dystrophin gene. For the first time in the world, we have shown that the biochemical method based on the radial model of the structure of the chromosome really allows mapping DNA loops observed on cytological preparations. This also confirms the radial model of the structure of the chromosome, on the basis of which our method of cutting loops was developed. Further, the possibility of observing identical DNA loops in the analysis of a number of nuclear halo preparations confirms the fact that DNA is statically organized into loops; in all cells, the same DNA fragments are attached to the nuclear matrix, and the sections between them form loops. We set up an experiment on actively dividing cells. Since the same DNA loops are found in all cells, it can be argued that the specific organization of DNA into loops separated by attachment zones is preserved in a series of cell divisions. This circumstance is extremely important, since it allows us to consider such an organization of DNA as one of the epigenetic mechanisms. Indeed, during the formation of loops, the positions of various regulatory elements and their targets can be fixed, facilitating their interaction or, conversely, excluding it.

DNA loops, chromosomal rearrangements and genome evolution

As we have said, the dystrophin gene recombination hotspots are in segments attached to the nuclear matrix. Additional studies have shown that recombination hotspots are also attached to the nuclear matrix and are present in a number of other genes, in particular those whose recombination is associated with the development of leukemias. It's hard to believe that this was just a coincidence. Most likely, it is the constant contact of DNA with topoisomerase that causes the appearance of "hot spots" of chromosomal rearrangements. Topoisomerase II can directly participate in illegitimate recombination. It is even more likely that the double-strand breaks introduced by it in DNA, under certain conditions, can stimulate inaccurate repair of these damages.

It is known that the repair of double-strand breaks in the DNA of higher eukaryotes often leads to various recombination events. The possible role of topoisomerase II as an inducer of chromosomal rearrangements is indicated by numerous data that the use of inhibitors of this enzyme in tumor chemotherapy often causes secondary leukemia. The cells of these leukemias are characterized by various large-scale chromosomal changes, most frequent at sites of cleavage by DNA topoisomerase II. It is important to note that the sites of loop attachment on the DNA molecule are located quite far from each other, but inside the nucleus they can be in close proximity. Illegal recombination between such regions will lead to the loss or displacement of extended regions of the genome, which, in turn, may be an important factor in the evolution of the genome.

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The length of the DNA of a diploid set of human chromosomes is approximately 174 cm, the average length of the DNA of one chromosome is 5 cm. In the nucleus, the length of one chromosome is 0.5 - 1 micron. Such packing of the DNA double helix is ​​explained by its further sequential compaction.

Rice. 12. A-, B-, C- and D-forms of DNA

(A. S. Konichev, G. A. Sevastyanova, 2005, p. 90)

1. Nucleosomal level. The nucleosome is a DNA - histone complex that looks like a disk-shaped particle with a diameter of 11 nm. Nucleosomes were first described in 1974. A. Olins and D. Olins. Each nucleosome consists of a protein core or octamer and 2 turns of a double-stranded DNA fragment (Fig. 13).

Rice. 13. Model of the nucleosomal core. A segment of DNA (146 base pairs) wraps around the protein core, making approximately 2 turns (1¾) around it. (S. B. Bokut et al., 2005, p. 52)

The protein core (core) contains a set of 4 pairs of histone proteins H2A, H2B, H3, H4. These are the most conserved proteins in any genome. They are almost the same in peas and in humans.

Nucleosomes are linked by regions of DNA (linker DNA) free from contact with the protein core.

The laying of the linker region of DNA (60-80 bp) and the connection of nucleosomes with each other proceed with the help of histone H1. The molecule of this protein has a central (globular) part and elongated "shoulders". The central part is attached to a specific area on the surface of the core, elongated "shoulders" connect neighboring nucleosomes. In this case, DNA is wound around neighboring cortex each time in the opposite direction (Fig. 14).

Nucleosomes can be isolated by short-term treatment of chromosomes with deoxyribonuclease enzymes. In this case, the docking sites of nucleosomes are split. The human genome contains 1.5 x 10 7 nucleosomes.

The nucleosome level increases the packing density of DNA by 7-10 times. (Fig. 14, 20)

Fig.14. Nucleosome fibril model.

2. Nucleomeric level. Further compaction of DNA in chromatin is associated with the formation of nucleosome complexes (Fig. 15, 20). A compact chromatin fibril is formed, built either as a solenoid (spiral type of packing) or as a nucleomeric type (4-12 nucleosomes form a globule).

Nucleomeric folding of chromatin contributes to the shortening of the DNA strand by about 6 times, and both levels lead to DNA compaction by an average of 50 times (42-60).

3. chrome level.

The next stage of DNA compaction is associated with the formation of loop-like structures, which are called chromomeres (Fig. 16). In this case, two ways of packaging DNA with the help of non-histone proteins are possible:

Rice. 16. Chromomeric type of chromosome packing.

The nucleosome thread is divided into sections of 20 - 80 thousand pairs of nitrogenous bases (on average - 50 thousand). In places of breakdown there are molecules - globules - non-histone chromosomal proteins. DNA-binding proteins recognize globules of non-histone proteins and bring them closer together. The mouth of the loop is formed. The average length of the loop (300-400 nm) is similar in various organisms (Drosophila and humans) and includes approximately 50 thousand bases. Such a looped structure is called an interphase chromonema.

Chromatin of the “lampbrush” type is interphase euchromatin (Fig. 17.). It is believed that the loops have connections with proteins of the chromosome framework, nuclear matrix and lamina proteins.

Rice. 17. Fragments of lampbrush chromosomes from the nucleus of a newt oocyte.

You can see sections of DNA forming loops from the central axis. (S. Gilbert, 1993, v. 2, p. 186)

Fibril shortening at this level occurs on average 25 times, and at all 3 levels by 1000-1500 times.

4. Chromoneme level. During cell division, further compaction of chromosomes occurs - the formation of larger loops from the chromomeric fibril. On the surface, packed DNA molecules carry many proteins that form a kind of sheath. If this sheath is removed, then under an electron microscope one can clearly see that each chromatid is built from chromatin loops extending from the central axis. The diameter of such a package is 700 nm (Fig. 18).

Fig.18. Chromonemal type of chromosome arrangement.

5. Chromosomal level. Further compaction of chromosomes is provided by the looped laying of the chromonemal thread (Fig. 19.), which reduces their length by about 10 times.

Fig.19. Chromosomal type of styling.

At this stage, loops with the same organization are combined, blocks or minidisks are formed. Approximately 20 loops are involved in the formation of one minidisk. Thus, due to several levels of compaction, the DNA length is reduced by about 10,000 times. To chromosome condensation from the decondensed state is not spiralization, but very complex compactization complex associated not only with changing their linear dimensions, but also with regulation their work during the life of the cell. (Fig. 20)

In addition, the compactization of the chromosome is the most important process associated with the accurate transmission of hereditary information to the next generation.