They participate in restoring the original DNA structure. Molecular composition of the cell. b. Repair by replacement of modified residues

Total information

As discussed in Chapter 6, cell membranes provide many functions under normal conditions. The plasma and intracellular membranes either function independently - mainly due to integral proteins located on both sides of the membrane, or participate in the cell functions associated with them (due to control by receptor and transport proteins), as well as the functions of the structural components of the cell formed from other proteins, lipids, sugars and microelements.

In general, cell damage is accompanied by disturbances in the genetic, biochemical and physicochemical processes occurring in it and is associated with transformations of macro- and micromolecules, including: polynucleotides and nucleotides, polypeptides and peptides, polysaccharides and monosaccharides, lipids and their components (phospholipids, sphingolipids, fatty acids, cholesterol), as well as movements of metal ions and other chemical compounds, free radicals, electrons, atomic and attomolar structures.

That is why the protective and restorative (repair) enzyme systems are so important for the cell in case of various types of damage. Let us consider these systems, taking into account the data on the structural organization, causes and mechanisms of cell damage presented in previous chapters of the manual, as well as data on the main forms of cell death.

PROTECTIVE AND RESTORATIVE

ENZYME SYSTEMS OF THE ORGANISM

As you know, the main protective and regenerative systems of the body include the nervous, endocrine and immune systems (see chapters 13-15). Then (in order of importance) come the detoxifying systems of the internal organs (liver, spleen, kidneys),

tissues (skin and mucous membranes) and, finally, cellular protective and regenerative systems, including: cytochrome P 450 systems, glutathione-dependent enzymes, superoxide dismutase (SOD), catalase, plasmalogens, peroxidases and phospholipases (in particular phospholipids) and other systems associated with restoration of structure-forming components of the cell. Numerous DNA repair systems and the still little studied enzymatic protective systems of biological fluids of the body (blood, lymph, cerebral fluid, gastric and intestinal juice) deserve special attention. All of these protective and regenerative systems of the body are based on the action of genes united in single gene networks (see Chapter 2).

First of all, let's look at the most well-known cell defense systems.

Xenobiotic detoxification systems

Systems of inactivation (detoxification) of substances or xenobiotics foreign to the body are controlled by environmental genes encoding the synthesis of numerous enzyme proteins that metabolize (degrade and detoxify) and remove xenobiotics from the body.

Mutations in detoxification genes often cause a hereditary predisposition to serious diseases that affect various body systems and individual organs. For example, the connection between the clinical manifestations of cystic fibrosis caused by a major mutation (CFTR) and the state of the genes of the detoxification system has been well studied. The main manifestations of this disease are severe chronic obstructive pneumonia, leading to the premature death of sick children, who, as a rule, do not live to the age of twenty. In turn, mutations in the detoxification genes themselves often predispose to the development of lung diseases: bronchial asthma, chronic obstructive bronchitis, cancer, emphysema and other pulmonary pathologies.

It has also been suggested that allelic variants of detoxification genes may influence clinical manifestations in cystic fibrosis. This assumption was confirmed in the work of M.A. Bakay et al. (1999). It turned out that patients with a mixed form and severe course of cystic fibrosis were either homozygous for the “zero” allele of the glutathione transferase M1 gene (GSTM1 0/0) -

the enzyme of the second phase of xenobiotic detoxification, or had a slowly expressed S-allele of the gene of the first phase of detoxification - microsomal epoxyhydrolase (mEPXH).

This work also noted a significant predominance of the homozygous state for the S (or S/S) allele in the mEPXH gene in patients with chronic respiratory diseases.

Considering the clear correlation of pulmonary pathology with the “slow” allele of the mEPXH gene and the “null” allele of the GSTM1 gene, we concluded that in patients with cystic fibrosis associated with the corresponding distribution of alleles (mEPXH S/S and GSTM1), pulmonary pathology develops most often and proceeds especially unfavorable. For this reason, the authors considered it appropriate to clarify not only the nature of the mutation in the cystic fibrosis gene, but also to determine in patients who carry unfavorable alleles of the mEPXH S/S and GSTM1 genes. In other words, this work shows a clear influence of certain alleles of the DQA1 genes of the HLA locus on the severity of cystic fibrosis and chronic respiratory diseases.

Detoxification with cytochrome P 450

In the membranes of the ER cells of the liver, as well as other organs and tissues, there are monooxidase enzyme systems that provide biosynthetic and detoxification processes to neutralize (transform) xenobiotics (drugs and man-made compounds - procarcinogens) in the first phase of detoxification. These systems are formed from hyperfamilies of cytochrome P 450 or monooxygenases that metabolize a wide range of exogenous and endogenous substrates.

As of January 2006, 1174 proteins and the corresponding number of genes encoding them, related to cytochromes P 450, were identified in the genomes of humans and animals (Lisitsa A.V., 2007). Moreover, the number of known chemical compounds that interact with enzymes of the cytochrome P 450 system was 1708, including 1223 substrates, 115 inducers and 484 inhibitors.

About 50 forms of cytochromes P 450 have been isolated in the human body (Helson, 1998), which participate in the reaction of monooxygenase catalysis and play a leading role in it due to the ability to selectively bind to the substrate. The functioning of cytochromes P 450 is determined by their interaction with partner proteins. Cytochrome P 450 enzymes close the electron transport chain and use the resulting redox equivalents to oxidize the substrate. As

The protein partners of cytochrome P 450 can be either several proteins, for example NADPH-cytochrome P 450 reductase and cytochrome b5, as well as adrenodoxin reductase and adrenodoxin, or it can be only one protein, for example reductase.

Among the human genes encoding cytochrome P 450 enzymes, the aromatase gene CYP19 was isolated, responsible for the synthesis of an enzyme that catalyzes the transition of androgens to estrogens in various tissues, including breast tissue. This gene has 11 DNA polymorphisms associated with increased or decreased expression (alleles: TTTA 7, TTTA 11 TTTA 12, etc.).

Another gene of this system, the CYP1A1 gene, encodes cytochrome P 450 1A1, which metabolizes hydrocarbons from tobacco smoke and also has its own polymorphisms (T623C, A4489G).

The CYP17 gene encodes cytochrome P 450 c17alpha. It is involved in the biosynthesis of sex steroids.

It should be noted that the localization of cytochromes P 450 in liver microsomes ensures that they receive electrons from the flavoprotein protein (cytochrome P 450 reductase), and the cytochromes P 450 themselves are the active form of the hemoprotein protein. In this case, the stability of this protein is ensured by the lipid bilayer of the membrane, consisting of phosphatidylcholine or a mixture of microsomal phospholipids.

It has been established that when cytochrome P 450 interacts with a xenobiotic, it is inactivated, during which it passes from the active form of cytochrome P 420 to the inactive one. In addition, this inactive form can be obtained by incubating isolated liver microsomes at 37 °C.

In the “xenobiotic-cytochrome P 450” reactions, all xenobiotics act as either exogenous substrates (carcinogens, drugs, food additives, toxins, poisons) or endogenous substrates (fatty acids, prostaglandins, steroids, cholesterol). The molecules of these substrates bind to cytochrome P 450 molecules in the ER membranes, causing a chain of lipid peroxidation reactions and other changes. At the same time, they can act both directly and indirectly. In the second case, obligate and facultative xenobiotics are distinguished among them.

Obligate xenobiotics, for example, phenobarbital, have a direct toxic effect on hepatocytes (the effect is dose dependent), which leads to hepatomegaly due to the induction of liver enzymes.

In turn, the xenobiotic cortisone causes fatty liver or steatosis through transformation into highly toxic intermediates such as salicylates and polycyclic hydrocarbons.

In addition, the direct effect of obligate xenobiotics or their metabolites causes disruption of bilirubin metabolism (at all stages of its synthesis from heme to excretion into the bile ducts), dilatation of sinusoids and occlusion of liver veins, which leads to necrosis and, less often, apoptosis of liver cells.

Facultative xenobiotics cause immune-mediated reactions underlying idiosyncrasies or intolerances to compounds, such as allergic reactions to drugs.

The cytochrome P 450 molecule itself is damaged due to two mechanisms of damage: the protein itself by free radicals and the lipid environment of the protein in the membrane.

In general, the mechanism of detoxification of xenobiotics in the liver with the participation of cytochrome P 450 includes three phases.

First phase- contact of the xenobiotic with ER enzymes (microsomal fraction of hepatocytes), monooxygenases, cytochrome c-reductases, reduced NADP (as a cofactor) and cytochrome P 450. During contact, modification of the xenobiotic occurs with the formation (release) of functional groups.

Second phase- biotransformation or conjugation (combination) of a xenobiotic molecule with endogenous molecules with the participation of two enzymes: UDP-glucuronyltransferase and glutathione-S-transferase, providing detoxification or reduction of toxicity with accelerated elimination of the xenobiotic (see below). In this phase, microsomal glutathione-transferase is directly associated with the cytochrome P 450 molecule, which contributes to the rapid inactivation of intermediate metabolic products or reaction metabolites of the xenobiotic.

Third phase- active transport and excretion (evacuation) of xenobiotic products transformed with the help of cytochrome P 450 with bile and urine.

Thus, inactivation of cytochrome P 450 is an indicator of damage to ER membranes in the liver. At the same time, the rate of inactivation of cytochrome P 450 can be used to judge the restoration or repair of damaged cell membranes.

Detoxification of phospholipid hydroperoxides

The phospholipid hydroperoxide (LOOH) detoxification system includes three glutathione-dependent enzyme complexes that perform two-electron reduction of LOOH: glutathione peroxidase, phospholipid peroxide-glutathione peroxidase, and selenium-containing glutathione transferase, type alpha (-SH-S-) or GST complex. These enzyme complexes are classified as antioxidants - quinolam(enolam), are present in all cells, tissues and organs, interact with vitamins E, C and ubiquinol (coenzyme Q) and are universal systems for binding active metabolites. All three glutathione-dependent antioxidant systems belong to enzyme redox system of glutathione(FRSG), which is involved in LPO and proliferation of immunocompetent cells. GFSH controls the division and activity of prooxidants, inhibits the free radical stages of peroxidation, destroys (non-radical) peroxides and interacts with non-peroxide products of peroxidation.

The properties of the redox system are determined by the state of balance: “oxidation-reduction” (see Chapter 9).

The level of activity of glutathione-dependent enzymes in different cells and tissues reflects the state of the entire antioxidant system, since these enzymes are necessary to protect against the aggression of ROS, chain free radical reactions and intensification of processes

The induction of glutathione-dependent enzymes is characterized by the “redundancy” of their activity and balance in relation to the oxygenation of cells, tissues and organs.

A special role belongs to the GST complex, which includes 21 enzymes, of which 16 are truly cytosolic. They are grouped into 6 subfamilies (classes): alpha, mu, omega, pi, theta and zeta. Each class is a dimer consisting of two equal or different subunits (with their own active sites) that act independently of each other. Each subunit has 2 domains connected by a short linker chain of 6 amino acid residues.

Each of the six classes of enzymes is encoded by gene clusters.

Alpha class- the gene cluster is located in the 6p12 locus, containing the genes GSTA1, GSTA2, GSTA3, GSTA4 and GSTA5 and 7 pseudogenes

new Genes of this class are involved in the metabolism of bilirubin, heme and steroid hormones. The GSTA1 and GSTA2 genes are expressed in all tissues. The GSTA3 gene was isolated from 8-9 week placenta. The GSTA5 gene has not been isolated from tissues.

Mu class- the cluster is mapped to locus 1p13.3. Includes 5 genes: GSTM1 gene (represented by three allelic variants: A, B and O; expressed in liver and blood cells); GSTM2 gene (only in muscles); GSTM3 and GSTM4 genes (testicles and brain); GSTM5 gene (lungs, brain, heart, testicles), as well as 2 pseudogenes.

Omega class- the cluster consists of two genes mapped to locus 10q25.1. Of these, the GSTO1 gene encodes a unique housekeeping enzyme that removes S-thiol radicals formed in response to oxidative stress. The second gene is GSTO2 (little studied). Transcripts of these genes are widely represented in all tissues. Their maximum expression is observed in the liver, skeletal muscles and heart; minimal expression occurs in the lungs, brain, and placenta.

Pi class- is represented by one GSTP1 gene, mapped to the 11q13 locus, and a GSTPP pseudogene, mapped to the 12q13-q14 locus. The GSTP1 gene has 3 allelic variants associated with polymorphism of nucleotide sequences in codons 105 and 114, related to the active centers of enzymes. This gene is an inhibitor of protein kinases involved in cell proliferation and apoptosis.

Theta class- its cluster includes 2 genes (GSTT1 and GSTT2), mapped to the 22q11.23 locus. The GSTT1 gene exists in two allelic variants (active and null). The GSTT2 gene has been poorly studied.

Zeta class- is represented by one gene GSTZ1, mapped to the 14q24.3 locus. The gene is expressed in liver cells and, to a lesser extent, in skeletal muscle and brain cells. Participates in the exchange of phenylalanine and tyrosine.

It should be noted that the enzymes of the GST complex, synthesized by all of these gene classes, function based on numerous mechanisms, including:

Catalytic inactivation of a xenobiotic through its connection with glutathione or the substitution path at electrophilic carbon atoms (halogen and nitroalkanes), nitrogen (trinitroglycerin), sulfur (thiocyanates and disulfites) and phosphorus (methyl parathionine);

Non-catalytic conjugation (binding) with the substrate; the classic substrate is 1-chloro-2,4-dinitrobenzene; this path is also typical for the conjugation of oxyarenes, alkene epoxides, nitronium and carbonium ions, as well as free radicals;

Reduction of organic hydroperoxides (lipid and DNA hydroxyperoxides) to alcohols through the expression of GSH peroxidase activity or reduced glutamine;

Isomerization of prostaglandins and steroids;

Participation in the metabolism of other exogenous compounds.

Reduced glutathione(GSH) refers to low molecular weight thiols that dominate in most proliferating cells. This tripeptide (L-gamma-glutamyl-L-cysteinylglycine) participates in the glutamyl cycle and neutralizes various toxic compounds by converting them into thioesters (this attacks nitrogen, oxygen, sulfur and carbon atoms). During further metabolism, glutathione conjugates are converted into mercapturic acids (mercaptans) and are excreted from the body (at a rate of about 0.1 mmol per day).

The importance of GSH can be traced in cancer patients, in whom the concentration of free glutamine depends on the properties of cytostatics, which deplete its supply, causing lysis of tumor cells. In addition, during cell proliferation and tumor growth, the decrease in the concentration of thiols and disulfites depends on maintaining the balance of metabolism, the synthesis of glutathione-dependent enzymes and the removal of phospholipid hydroperoxides.

LOOH detoxification proceeds according to schemes that include:

Peroxide-Ca 2 + -stimulated PLA 2 and glutathione peroxidase along the “hydrolysis-reduction-repair” pathway;

PL-peroxide-glutathione-peroxidases and phospholipases A 2 along the “reduction-hydrolysis-repair” pathway.

Both schemes lead to the reacylation of lysophospholipids.

At the same time, under conditions of an imbalance between endogenous oxidants and antioxidants, an excess of ROS and subsequent oxidative stress may occur. The mechanism of such stress, called “oxidative stress signaling,” accompanies LPO reactions in response to dangerously high or low levels of oxidants.

Oxidative stress signaling

With the help of oxidative stress signaling (OSS), the cell produces an excess of antioxidants and activates one or several classes of enzymes: reduced glutathione (see above), heme oxygenase-1, glutathione peroxidase, catalase, SOD and ferritin.

OCC has been shown to stimulate the expression of genes that control apoptosis, cytoprotection, and other cellular effects.

OCC involves protein kinase C (PK C), MAPK, hydrolases, as well as phospholipases C (PL C) and A 2 (PLA 2), activated by Ca 2 + ions. Early mediators of these enzymes are LOOH (see above), which are considered “mimetics” of the effects of growth factors (TNF-alpha), cytokines and other agonists, indicating a role for oxidative derivatives as second messengers (see Chapter 8).

For example, oxidative activation of phospholipases through the release of arachidonic acid initiates the formation of eicosanoid lipid mediators and lysophospholipids, which act as compounds that produce direct effects or are metabolized to PAF and other effectors.

After the action of oxidants, which particularly damage cell membranes, the fate of the cell is twofold: either survival or death through apoptosis or necrosis - this depends on the severity of “oxidative stress”. For example, using endo or exogenous peroxides, apoptosis can be triggered in leukemic cells. A possible mechanism of activation by cytosolic phospholipase (see Chapter 9), which affects arachidonoyl phospholipids, includes PKC-catalyzed phosphorylation of the enzyme and its translocation in the membrane (through MAPK activation). This mechanism depends little on Ca 2+ and, therefore, is activated only at relatively low LPO levels.

Superoxide dismutase system

The SOD enzyme catalyzes the reaction between two superoxide radicals to form hydrogen peroxide (H 2 O 2) and molecular oxygen (O 2). This reaction is called dismutation reactions(see chapters 6 and 9).

The SOD 1 gene is localized on chromosome 21, and this localization has been discussed many times in the literature to explain the causes and mechanisms of the development of Down syndrome (as a triple dose of the gene). However, this has not been confirmed. It turned out that in the mitochondria of normal cells, which serve as a source of ATP and ROS, the permissible

the level of the latter is maintained by a protective enzyme system, which, along with SOD, also includes cytochrome oxidase, glutathione peroxidase and some other enzymes.

Antioxidant role of blood

As noted in Chapters 8 and 9, signaling mechanisms play an important role in the regulation of membrane enzyme activity. Most of these mechanisms are realized through the blood, which has its own antioxidant activity and is in direct contact with the structural components of the walls of blood vessels, mainly represented by endothelial cells.

The regulation of enzyme activity by structural and functional elements of the blood can be demonstrated by the example of “tissue factor”, or transmembrane glycoprotein, found in atherosclerosis in the adventitia of blood vessels and the internal region of the atherosclerotic plaque. This factor is expressed on the membranes of monocytes and macrophages and is involved in inflammation and plaque destabilization; it is especially sensitive to the action of inhibitors synthesized by endothelial cells of blood vessels during signaling.

It is important to note that oxygen is transported through the blood, the bactericidal effect of phagocytes is carried out, and metal ions of variable valence, which are catalysts or inhibitors of oxidation-reduction processes, move. At the same time, blood, to a certain extent, is the limiting link in the antioxidant defense system, since compared to the intracellular contents it contains few high-molecular antioxidants, which enhances oxidative reactions and does not favor reductive reactions.

Mechanisms of cell membrane restoration

Biochemists have long suggested that cell membranes adapt to damage. This is supported, for example, by data on the restoration work of enzymes due to reactions carried out by PC C, MAPK and mitochondrial ATP-dependent calcium channels. In particular, the opening of calcium channels in mitochondrial membranes causes their depolarization, reducing excess accumulation of Ca 2+.

It is this mechanism that promotes cell regeneration during myocardial infarction.

Restoration of enzyme activity has been experimentally demonstrated in vitro damaged microsomal membranes in the liver of rats when PC or a mixture of PC and lysoPC was added to them. The addition of these phospholipids restored the activity of glucose-6-phosphatase, Ca2+-, Na+-, K+-ATPase, stearoyl-CoA desaturase, lipase, UDP-glucuronyltransferase, phenylalanine hydroxylase, and cytochrome b5 reductase.

Similar results were obtained for brain sphingomyelinases, cardiac mitochondrial beta-hydroxybutyrate dehydrogenase, and bovine erythrocyte acetylcholinesterase.

In connection with these data, it was suggested that the restoration of the activity of membrane proteins by phospholipids is nonspecific, since most enzymes do not require certain types of phospholipids, and therefore different types of the latter (or mixtures thereof) can perform the reducing function for them.

The very fact of the participation of membrane phospholipids in restoring the activity of enzyme proteins is interesting. At the same time, to maintain (and possibly restore) the activity of membrane enzymes, the fatty acid composition of phospholipids added to cells is important. For example, this was shown for Ca 2 + -ATPase of the sarcoplasmic reticulum, when replacing its own phospholipids with DPPC (phosphatidylcholine with a high content of saturated fatty acids) reduced activity by 8 times compared to the partially delipidated enzyme, and when replacing its own phospholipids with dioleyl-PC the reaction rate, on the contrary, increased sharply.

In addition, the importance of high mobility of phospholipid hydrocarbon chains for restoring enzyme activity and the presence of a certain surface charge on membranes has been noted.

Similar results were obtained for:

Acid phospholipids (PS, PI and FA) in their action on laryngeal myosin phosphatase;

Anionic phospholipids (PS) - when acting on 5-monodehydrogenase of the human placenta.

Thus, at present we can talk about the endless variety and significance of not yet discovered, but apparently actually existing mechanisms for restoring structural and functional damage to cell membranes.

Mutagenesis inhibitors

As stated in Chapter 5, mutagenesis is the process of the formation of mutations in hereditary material, induced by the action of various mutagenic factors.

Back in the middle of the 20th century. A. Novik and L. Szilard showed that apurinic ribonucleosides reduce the level of spontaneous and induced mutations in E. coli, which allowed them to identify a new class of chemical compounds called antimutagens.

At the end of the 20th century. S de Flora and S. Ramel divided antimutagens into two classes: extracellular (dismutagens) and intracellular.

Extracellular antimutagens include three subgroups.

Inhibitors of the absorption of antimutagens and their precursors (aromatic amino acids, fatty acids, etc.). They prevent the penetration and accelerate the removal of mutagens from the body.

Inhibitors of endogenous formation of mutagens (ascorbic acid, tocopherol, phenols, fermented dairy products). They prevent or inhibit nitrosation reactions or alter the intestinal flora.

Deactivators of mutagens as a result of physical and/or chemical reactions (antioxidants, substances that maintain pH levels in biological fluids, and thiols).

Intracellular antimutagens also include three subgroups.

Metabolism modulators (thiols and phenols). They accelerate the transfer of mutagens to non-target cells and induce a detoxification mechanism.

Inactivators of reactive molecules. They interact with electrophiles, protect nucleophilic regions of DNA, and trap oxygen radicals.

Modulators of DNA replication and repair (sodium arsenite, vanillin, protease inhibitors, coumarin, thiols, cobalt chloride). They increase the accuracy of replication, increase the efficiency of repair, and inhibit repair errors.

As follows from this classification, the same compound can belong to several groups, for example thiols.

In addition to extracellular and intracellular antimutagens, B. Stavrik isolated in 1992 more than 25 antimutagens or chemopreventers contained in them from different types of food products. This group includes vitamins, fatty acids, calcium, carotid

noids, coumarins, dietary fiber, plant acids, selenium, flavonoids and chlorophyll.

Antimutagens of plant origin include green peppers, cabbage, mint leaves, onions, plant seeds, and apples.

The action of antimutagens is specific: it manifests itself with high selectivity and depends on the dose. In this case, some antimutagens may be inhibitors of mutagens, have the opposite comutagenic effect, or their effect may not appear at all. In particular, such antimutagens include vitamins C,

The possibility of protecting the cells of some tissues with the simultaneous potentiation of mutagenic effects in the cells of other tissues cannot be ruled out.

In general, the question of the effectiveness of exogenous and endogenous mutagenesis correctors still remains open.

In this regard, let us move on to consider the more well-known and widely discussed in the scientific and educational literature of the recovery mechanisms in a damaged cell.

Restoring DNA structure using repair mechanisms

As is known, cells have a restorative ability or the ability to correct and eliminate (repair) damage to the DNA molecule, restoring its original structure. Due to this, only a limited number of mutations are maintained during the process of replication, transcription and translation.

However, if the mutation is not recognized, the distorted information is transcribed into mRNA and expressed in the form of a defective protein. The consequences of such an event for the cell are often insignificant, but sometimes they are extremely undesirable, and this depends on the functions performed by the defective protein.

Currently, numerous repair mechanisms have been well studied, both known since the times of classical genetics (photoreactivation, repair of thymine dimers; excision repair; SOS repair), and those discovered in recent years (see below).

Summarizing the features of these mechanisms, we can conclude that the repair of various types of damage in the DNA molecule occurs in several stages:

First step- identification of damage and determination of its type;

second phase- this is the activation of enzymes that either directly transform the damage to its original state, or (if direct repair is not possible) cut out the damaged area, forming a gap.

In the latter case, two more steps are added:

third stage- this is the synthesis of a new section of the DNA molecule (to replace the damaged one);

fourth stage- this is the embedding of a new section into the gap.

Photoreactivation or repair of thymine dimers

Under the influence of UV irradiation, covalent cross-linking of two adjacent pyrimidines (two thymines) located in different strands of the molecule can occur in a DNA molecule. In this case, thymine dimers (cyclobutane ring) are formed, blocking DNA replication. During the repair of thymine dimers, the enzyme that “recognizes” them, photolyase, combines with them, forming a single complex. UVR activates this enzyme and the cyclobutane ring breaks to form two separate thymines. A. Kellner named this mechanism in 1949 photoreactivation.

Deamination (methylation) and mismatch repair

As stated in Chapter 6, during deamination adenine is converted to hypoxanthine, which forms hydrogen bonds with cytosine. Guanine is converted to xanthine, which forms hydrogen bonds with thymine. Thymine cannot be deaminated (the only nitrogenous base in DNA). When cytosine is metabolized, uracil is formed. If the deamination processes of these nitrogenous bases are disrupted, erroneous or incorrectly paired bases appear in the DNA molecule. So, instead of AT pairs, GT pairs are formed in the DNA chain, and instead of GC pairs, HA pairs are formed - these are pairing errors. They are eliminated using two mechanisms: GT-mismatchreparations And GA-mismatch-repairs respectively. Participants in these reparation mechanisms may include:

Protein products of four Mut family genes: H, L, S and U;

Helicase proteins;

DNA polymerases that have the property of “taking a step back” after placing the next nucleotide into the growing DNA strand and cutting out the last nucleotide if it is non-complementary to the nucleotide in the template DNA strand, i.e. capable of correcting

create pairing errors;

Adenosine triphosphatase (ATP);

Deoxynucleotide phosphatases (dNTPs).

It should be noted that the mechanisms mismatch reparations operate on the daughter strand and replace non-complementary bases only in it.

Shortly after replication ends, methylase enzymes add methyl groups to adenines in GATC (guanine-adenine-thymine-cytosine) sequences.

During the next replication, the DNA strands become distinguishable: the mother strand contains methylated adenines, and the adenines in the daughter strands are not yet methylated until the end of replication. Their methylation will begin only after the end of replication. Therefore, while adenines are not methylated, cells must have time to “repair” mismatches. Repair of mismatched bases can occur during DNA deamination, when the enzyme N-glycosylase recognizes the base, breaks the covalent N-glycosidic bond, and removes it. In addition, mismatch repair can occur during DNA methylation (see Chapter 7).

If the formation of classical “Watson-Crick” base pairs is generally difficult, then pairing errors (replication errors) can occur during replication and mismatches can also occur. To eliminate them, other enzymes are involved: first, the endonuclease breaks one DNA strand in places where AT or GC pairs have not yet formed, then phosphodiesterase cleaves off at these places of breaks those sugar-phosphate groups (AP sites) to which no bases are attached. The gaps that appear in the chain of molecules (one nucleotide in size) are filled by DNA polymerase I, after which the enzyme ligase stitches the ends of the DNA, restoring the original state of the molecule.

Alkylation and repair of O 6 -alkylated (methylated) guanine and O 4 -alkylated thymine

Alkylation - is the addition of alkyl side groups (methyl, ethyl, propyl or butyl) to the purine or pyrimidine bases of a DNA molecule by the action of a number of chemical mutagens (alkylating agents). For example, methylnitroso-guanite alkylates (methylates) guanine by adding a methyl group to it.

During repair of O 6 -alkylated guanine(About 6 meg) the methyl group is cleaved from guanine due to the oxygen associated with the sixth atom, and at this point the DNA structure is restored. This mechanism was discovered by I.A. Rapoport in 1944

It has been established that during alkylation, proteins are synthesized in cells - methyltransferases, which capture its methyl groups from the modified guanine, restoring the original DNA structure. Moreover, methyltransferases that have captured such groups can no longer get rid of them, which indicates that they do not belong to the class of enzymes that do not change during numerous reactions. In addition to this mechanism, there is repair of O 4 -alkylated thymine(O 4 -alT). Consequently, if we assume that for each act of direct DNA repair new protein molecules are needed, then in the event of damage by alkylating agents the cell is forced to organize their synthesis in the ratio: one molecule per one damage. Therefore, the processes of DNA damage formation and their repair are interconnected. Typically, thousands of such molecules accumulate inside the cell. For example, during one cell cycle that occurs in E. coli for 30 minutes, about 3000 methyltransferases can accumulate, capable of neutralizing 3000 DNA damages.

Apurinization and excision repair

Apurinization - this is the formation of AP sites in the nucleotide chain

As is known, each somatic cell, when functioning, loses approximately 10 thousand purines and pyrimidines per day, and as a result, apurine sites or sugar-phosphate groups without bases (AP sites) are formed in its DNA molecule. If AP sites were not corrected, there would be a disaster.

During the elimination of AP sites, insertase enzymes break the covalent N-glycosidic bond between the base and deoxyribose, and then direct insertion of purines occurs. This mechanism was discovered by T. Lindahl in 1979.

Causes of apurinization: increased temperature, change in pH, ionizing radiation.

If the cell cannot cope with damage to bases and nucleotides in the DNA molecule using the repair mechanisms listed above, then more complex repair mechanisms come into play, one of which is excision repair,

carried out by cutting out (excision) a damaged base in a nucleotide or a more extended section of the molecule.

Excision repair Damage to bases occurs with the help of DNA glycosylases, which recognize base damage resulting from methylation, oxidation, reduction, deamination, addition of formide groups and other reactions.

The DNA glycosylase family includes 11 types of enzymes that bind to substrates or damaged targets: ura- (contains uracil); hmu-(hydroxymethyluracil); 5-tC-(methylcytosine); Hx-(hypoxanthine); 3ntA-, type I (3-methyladenine) and type II (contains methyladenine, 7-methylguanine or 3-methylguanine); FaPy-(formidopyrimidine or 8-hydroxyguanine residues); 5, 6-HT or endonuclease III (5,6 hydrated thymine residues); PD-(pyrimidine dimers); thymine mismatch (contains a non-complementary GT base pair); substrate mutY (contains a non-complementary GA pair).

DNA glycosylases attach to the lesions and break the glycosidic bonds between the modified base and deoxyribose, resulting in the formation of AP sites.

The resulting AP sites are recognized by the enzyme AP endonuclease (capable of breaking the chain inside a DNA or RNA molecule). After the break appears, the enzyme phosphodiesterase comes into action, which cleaves from the DNA the sugar phosphate group to which the base is no longer attached.

As a result, a gap of one nucleotide in size appears in one DNA strand. Opposite the gap in the opposite DNA strand there is an intact nucleotide, and another enzyme (DNA polymerase I) inserts a complementary nucleotide into the gap, attaching it to the free 3" OH-end. This end of the DNA strand and the 5" end previously formed when the strand is broken are connected each other under the action of polynucleotide ligase. After this, the entire structure is considered to be completely restored: the incorrect base is removed, the sugar phosphate to which the base was attached is cut out, the gap is filled with the correct nucleotide, and the single-strand break is repaired.

Excision repair of damaged nucleotides

The excision repair mechanism is a more complex and more energy-consuming mechanism associated with cutting out not just the damaged base, but a significant section of the chain

DNA before and after damage. In Escherichia coli, such repair is carried out by a multienzyme complex of endonucleases encoded by three ultraviolet repair genes or uvr genes (A, B and C) and called the excinuclease complex (Fig. 50). This mechanism was described by R. Setlow and V.N. Soifer in 1970. It includes the following stages:

Damage recognition by uvr A and B proteins;

Bending of the DNA molecule and change in the conformation of the uvr B protein;

Making two single-strand DNA cuts on both sides of the damage using uvr proteins B and C;

Unwinding of DNA in the area between two cuts using the uvr D protein (helicase II);

Detachment of a fragment containing a defect of 12 nucleotides in length (12-mer) with the formation of a gap, which consumes the energy of one ATP molecule;

Filling up the resulting gap using DNA polymerase;

Joining of free ends of the sugar-phosphate backbone of DNA using DNA ligase.

A similar mechanism exists in humans. At the same time, its excinuclease complex consists of 17 proteins, filling the gap

Rice. 50. The mechanism of excision repair in E. Coli. (after Elliot W., Elliot D., 2002)

occurs with the participation of DNA polymerases O&E, and the section excised from the damaged DNA strand is 29 nucleotides long (instead of 12 in E. coli).

Repair of damaged nucleotides includes mismatch repair or repair of non-complementary (non-canonical) or non-Watson-Crick base pairs - this is the so-called mismatch repair (see above).

Repair of single-strand and double-strand DNA breaks

In addition to the repair mechanisms discussed above, mechanisms for repairing single-strand breaks (breaks in bonds between bases in one strand of a DNA molecule) and double-strand breaks (breaks in bonds between bases in two strands of a DNA molecule) are known.

Repair of single-strand DNA breaks

Repair of single-strand DNA breaks - this direct reparation. It is caused in response to the action of ionizing radiation and is ensured by the sequential action of enzymes: 3"-phosphodiesterase, DNA polymerase-b and DNA ligase (DNA polynucleotide ligase). The restoration of such a break occurs using an intact complementary DNA strand as a template.

Repair of double-strand DNA breaks

Double-strand breaks in the DNA molecule are the most dangerous type of DNA damage for cells. They usually lead to the development of genetic instability, the appearance of point mutations and chromosomal aberrations and subsequent cell death.

There are two known mechanisms for the repair of double-strand DNA breaks: non-homologous rejoining of broken ends and homologous recombination.

Nonhomologous reunification of broken DNA ends

Nonhomologous reunification of broken DNA ends occurs between ends of the molecule that have nonhomologous nucleotide sequences of different lengths or very short homologous regions. This repair mechanism is not infallible and often serves as a source of point mutations, since restoration of the original structure of the DNA molecule is possible only by reuniting the ends of the same strand. If this condition is not met, then chromosomal aberrations are formed: deletions, duplications,

inversions, insertions and translocations (see Chapter 5). In mammals, nonhomologous reunification occurs with the participation of the Rad50 protein, related proteins, Ku factors, DNA ligase IV and silencing factors.

Homologous recombination

The mechanism of homologous recombination has been studied in Drosophila melanogaster. It is assumed that it should exist in human cells, but convincing evidence of this has not yet been obtained. It is believed that during homologous recombination a gap is formed on one of the DNA strands, equal in size to the element removed from bacteria (P-element). Repair of the gap occurs with the participation of a copy of the P element located on the sister chromatid and used as a template for repair synthesis.

However, during replication, a single-stranded gap may form opposite the unrepaired site, and this situation cannot be corrected without error without the involvement of another DNA molecule.

Therefore, this mechanism provides recovery only in cases where both DNA strands are damaged.

Alberts proteins and their role in DNA replication and repair

In 1968, Alberts proteins, or SSB proteins, involved in DNA repair, were discovered. These proteins, due to the organization of their tertiary structure, have the ability to bind electrostatically to DNA. SSB proteins contain a cluster of positively charged amino acid residues, but their overall charge remains negative, due to which they have an increased affinity for single-stranded DNA.

If, due to disturbances in the secondary structure of double-stranded DNA, “molten sections” are formed in individual strands of its helix, then Alberts proteins bind to them, easily sit on them and “straighten” them. At the same time, the SSB proteins sitting on the complementary chains of the molecule do not allow them to “collapse”, since due to electrostatic interactions they have a powerful negative charge, showing affinity for each other and covering the “molten area” with a continuous layer - this stoichiometric amount of protein. In other words, these proteins do not denature the DNA molecule, but only fix its single-stranded state.

The participation of SSB proteins in the replication of the DNA molecule is absolutely necessary for the cell: they keep both template strands in a single-stranded state in the replication fork, protecting each strand from the action of nucleases and selectively stimulating the work of DNA polymerase (RNA polymerase does not use single-stranded DNA coated with SSB).

Thus, the role of SSB proteins in the repair of single- and double-strand DNA breaks has now been fully proven.

Post-replicative (recombination) DNA repair

In 1968, W. Rapp and P. Howard-Flanders discovered that bacteria treated with UV rays post-replicative repair or recombinational repair of a template DNA strand, which contained many uncut thymine dimers, and at the moment when the DNA polymerase leading replication reached the first dimer, it “frozen” at this point for 10 s, then moved beyond the thymine dimer (somehow in an incomprehensible way) and resumed synthesis behind the defect until it “stumbled upon” the next dimer. Thus, sections of the daughter strand contained gaps (not duplicated during DNA synthesis), and unhealed defects remained in sections of the matrix strand opposite the gaps. This was followed by post-replicative repair of damaged areas, in which rec A proteins, ligases and DNA polymerases took part. This mechanism resembled the mechanism of recombination: first, a rec A protein molecule attached to the gap zone, where, under its control, recombination occurred, during which a section of the complementary chain of the sister strand was transferred to the gap zone, the gap was built up during replicative synthesis, and ligase connected the ends of the new and old strands of molecules.

SOS DNA repair

SOS DNA repair is the last option for the DNA of a cell that has approached replication with damage that has not been repaired by all of the repair mechanisms listed above. In this case, the cell may die, since replication may “stalle” at the first unrepaired damage.

At the same time, the cell has an extremely risky mechanism designed for such purposes, called SOS DNA repair. This mechanism was first discovered in 1953 by J. Wagle and

named in 1974 by M. Radman W-reactivation(the ability for thymine dimers to “survive” until the next replication). During the SOS repair mechanism, the synthesis of proteins is induced that attach to the DNA polymerase complex and “harden” its work in such a way that the damaged complex becomes able to build a daughter DNA strand opposite the defective links of the matrix strand, and at the same time many errors appear on the daughter strand ( mutations). As a result, the cell is saved from death at this stage and can achieve mitosis, although there will be errors and a high risk of cell death.

It should be noted that the above mechanisms of DNA molecule repair relate mainly to the achievements of classical genetics. At the same time, in recent decades, thanks to the achievements of the international scientific program “Human Genome” (see Chapter 1), the general list of DNA repair mechanisms has been significantly supplemented by other previously little-known and new mechanisms.

Repairing a DNA molecule using mechanisms discovered in recent years

The repair mechanism involving the enzyme poly-ADP-ribose polymerase

The protein-enzyme poly-ADP-ribose polymerase (PARP) is one of the nuclear factors that first recognize DNA damage. This factor controls the launch of the DNA repair mechanism in living cells, starting from the site of DNA damage, and is part of a multiprotein complex (MP complex), which also includes the XRCC1 factor, DNA ligase III and beta-DNA polymerase.

The presence of PARP in this complex ensures the direction of all DNA repair participants to sites of DNA damage in vivo, which facilitates the restoration of the DNA molecule.

The factor XRCC1, which is part of the MR complex, conventionally refers to the “scaffolding”, individually interacting with each component of the complex as a supporting molecular structure. Since polyADP-ribosylation of this factor was discovered in vitro, it was suggested that PARP is able to regulate the activity of the MP complex by modifying the XRCO protein in vivo, which disrupts its interaction with other components of the complex. This assumption was supported by data that overexpression of the XRCC1 factor suppresses PARP activity in vivo.

Currently, this hypothesis continues to be studied; it is confirmed by the data that DNA ligase III, which is part of the MP complex, inhibits the activity of PARP in vitro, when its quantity exceeds the quantity of PARP.

Repair model with anti-recombination effect of the enzyme poly-ADP-ribose polymerase

In recent years, a model of DNA repair with the anti-recombination effect of PARP has been proposed (Satoh and Lindahl, 1992). According to this model, PARP interacts with DNA breaks and, together with poly-ADP-ribosylation reactions of neighboring proteins, specifically protects DNA ends from the action of nucleases and/or blocks the recombination process. This is confirmed by the example of experimental animals deficient in the PARP gene. It has been shown that the absence of PARP stimulates the recombination process and leads to an increase in the incidence of lymphoma formation in them. In addition, PARP can recruit DNA repair factors by modifying chromatin proteins.

DNA repair using methyltransferases

With the help of supporting enzymes cytosine-DNA methyltransferases (M-taz), it is possible to carry out de novo methylation of cytosine residues in DNA to form 5-methylcytosine (5-mC), as well as methylation of oligonucleotides containing mispaired bases.

DNA repair using helicases

Among the enzymes involved in DNA repair, much attention is paid to the group of DNA helicases (chylases). These are phylogenetically stable enzymes capable of separating the chains of a DNA molecule, converting it from a native state to a single-stranded one. Helicases are involved in all fundamental genetic processes with the separation of the parent strands of the DNA molecule or processes based on such separation: replication, transcription, recombination, repair, chromosome segregation, processing and splicing of pre-mRNA, transport of mRNA into the cytoplasm and its translation on ribosomes (see. chapters 2 and 3). During these processes, chylases provide access to the single-stranded sections of the DNA molecule they open for the action of other enzymes. Chylases differ from each other in the directions of activity (3" - 5" and 5" - 3") of the DNA chain, preferential affinity for the 3" and 5" ends of the DNA chain, and affinity for cofactors - nucleotide triphosphates. These energy-dependent fer-

ments consume energy generated during the hydrolysis of nucleotide 5"-triphosphates (usually ATP) and act in the presence of Mg 2 + ions. When carrying out any genetic process, chylases, as a rule, combine into complexes.

Based on their affinity for the substrate, chylases are divided into groups: DNA helicases, RNA helicases, and helicases operating on hybrid RNA/RNA molecules. The former are involved in the initiation of transcription, recombination, repair and chromosome segregation. The latter are involved in ribosome biogenesis, mRNA transcription, pre-mRNA splicing, maturation and transport of mRNA into the cytoplasm and its translation on ribosomes.

Chylases are classified by activity (the ability to move along a DNA molecule, dividing it into separate chains), the presence of 7 specific amino acid sequence motifs that are inherent in the entire family of chylases, but are not required for individual ones. These motifs are designated by numbers: I, Ia, II, III, IV, V and VI (they provide connections between chylases and DNA and coordination of movement during catalysis). Although these motifs are specific to chylases, they are also found in proteins that use nucleotide triphosphates as a cofactor (like chylases). Mutations of these motifs cause a deficiency in ATP-dependent chylase activity. Along with chylases, proteins with motifs that do not have chylase activity have been isolated. There were also 7 of them, and they got the name protein candidates for chylases. It is believed that such proteins should also be involved in the regulation of intracellular genetic processes.

In addition, 2 mechanisms for the movement of chylases at different speeds along the nucleotide chain of a DNA molecule were identified: an actively rolling model and a gradually sliding model.

First mechanism is carried out by a DNA helicase dimer, which in one cycle (rotation of the molecule) separates a certain number of bp.

Second mechanism carried out by a DNA helicase monomer moving at a certain speed.

The number and properties of genes encoding helicases in the human genome have not been fully determined, but some genes have been well studied. These are helicase genes belonging to the TFIIH and RecQ families. Moreover, the first family is the main transcription factor, consisting of nine subunits, including two chylases (XPB and XPD), 4 proteins (p62, p52, p44 and p34), the SAC complex, the

activating kinase (CDK-activating-kinase), and 2 cyclins (H and Cdk7).

TFIIH has ATP-dependent helicase and kinase activities, which are controlled by the XPB and XPD subunits, respectively. Kinase activity is also provided by the SAC complex.

It has also been shown that TFIIH is involved in DNA repair (“recognizes” a DNA section of 20-30 bp in length containing damage and cuts it out) and serves as a transcription factor (interacts with the promoter region, unwinding a DNA section of 11-13 bp in length. n. around the transcription initiation site).

Repair of nonsense mRNA transcripts

Approximately one third of inherited diseases and many forms of cancer are caused by nonsense transcripts or frameshift mutations(see chapter 5). As a result of these mutations, PSCs arise and defective proteins appear.

At the same time, there are systems in cells that recognize and destroy most nonsense transcripts. Two such (universal for eukaryotes) systems are called: NMD and SMD (see Chapter 7).

Concluding the consideration of cellular DNA repair systems, it should be noted that the restoration of defects that arise in the DNA molecule as a result of exposure to mutagenic factors is the most important property of all living organisms.

Among the increasing number of DNA repair mechanisms are mechanisms simple, triggered immediately after damage to the DNA molecule, and complex, extended over time and requiring the synthesis of a number of enzymes. The latter include mechanisms associated with different stages of the cell life cycle, as well as saving the cell in the SOS order or the order of introducing new mutations in the DNA molecule. All restoration mechanisms have common pathogenetic features and are closely intertwined with the processes of carcinogenesis, mutagenesis, teratogenesis and aging of cells and organisms.

DISEASES OF THE GENOME

Diseases of the genome (genetic instability) are represented in all areas of molecular medicine. Since the times of classical genetics, the most famous of them are reparation diseases

In Russia in the 80-90s of the XX century. DNA repair diseases were studied under the name diseases of genetic (chromosomal) instability, which corresponded to their leading feature - increased fragility of chromosomes.

Currently, the range of these diseases has expanded significantly. Along with classical DNA repair diseases, such as ataxia-telangiectasia (AT), Fanconi anemia (AF), xeroderma pigmentosum (XP), Hutchinson-Gilford progeria (HG), Bloom syndrome (BS), the following groups of diseases belong to this class :

Autoimmune diseases: systemic lupus erythematosus, scleroderma, rheumatoid arthritis, etc.;

Hereditary enzymopathies: Knapp-Komrover, Lesch-Nyan, Pendred, etc.;

Chromosomal syndromes: Down, Klinefelter, Patau, Shereshevsky-Turner and Edwards; retinoblastoma caused by deletion of chromosome 13 (13q14);

Monogenic diseases: Friedreich's ataxia, Darier-White disease, Gardner syndrome, basal cell nevus, Marfan, Rothmund-Thomson, Cornelia de Lange, testicular feminization, photodermatosis, etc.;

Polygenic diseases: psoriasis, systemic sclerosis, etc. Genetic instability also manifests itself in practically

healthy carriers of balanced chromosome rearrangements, for example, with Robertsonian-type translocation between chromosomes

Common clinical manifestations of genomic diseases are: pronounced neurological manifestations, decreased life expectancy, symptoms of premature aging, and an increased incidence of malignant tumors.

First of all, let's look at examples of classical genome diseases, or DNA repair diseases.

Monogenic diseases associated with increased photosensitivity and impaired DNA excision repair

The most famous disease of this class is PC. Its frequency in the population is 1:40-250 thousand people. PC is first illness DNA repair described in humans. It is heterogeneous: it has 7 complementation groups - A, B, C, D, E, F and G. In particular,

Group A is widespread in Japan, where it accounts for approximately 20% of all patients with PC, and is associated with a defect in the post-replicative repair system (ChRta1). Complementation groups B and C are common in European countries, and groups D, E, F and G are common in other countries of the world.

The group A PC gene is localized at locus 9q34.1. Its product is a DNA-binding protein that has two zinc finger motifs (see Chapter 8).

The group B PC gene is mapped to the 2q21 locus; its protein product is a helicase, which is part of the TFIIH transcription factor (see above).

The group C PC gene is mapped to chromosome 3 and encodes the p125 protein. The function of this protein has not been fully determined, although it is known that, together with the p58 protein, it is involved in the restoration of repair activity.

The group D PC gene is mapped to the 19q13.2-q13.3 locus; its protein product (like the product of the group B PC gene) is characterized by helicase activity. It is believed that both gene products are two subunits of the same TFIIH factor.

The group F PC gene is mapped to the 16p13.13 locus; it produces an endonuclease that cuts DNA from the 5" side.

The G group PC gene is mapped to the 13q33 locus and also produces an endonuclease, but cuts DNA from the opposite 3" side.

The main symptoms of this disease are: high sensitivity to the action of ultraviolet radiation, pigmentation, dryness, ulceration and scarring of the skin. Patients develop cancer of the skin and mucous membranes (melanoma, carcinoma).

In every second or third case, neurological symptoms are expressed (associated with early apoptotic death of neurons).

In recent years, it has been established that 3 out of 7 PC complementation groups (groups B, D and G) can manifest themselves as genocopies of another DNA repair disease - Cockayne syndrome (CS), caused by defects in the endonucleases of the excision repair system.

In such cases, patients with PC can have common clinical signs with MC, including increased sensitivity to ultraviolet radiation. Therefore, the diagnoses in such patients are designated as PKV/SK, PKD/SK, PKG/SK.

At the same time, SK is second disease(after PC), described as excision repair disease. This syndrome is manifested by dwarfism (with normal levels of growth hormone), calcification of the skull bones, atrophy of the optic nerves, deafness and accelerated aging. In this syndrome, two complementation groups have been identified: A and B. The CK gene of group A is localized in the 5p12-p14 locus, it encodes a protein that is part of the TFIIH transcription complex (see above). The group B CK gene is localized in the 10q11.2 locus; it encodes a protein that has common nucleotide sequences with a factor that controls transcription and repair in E. coli, and in humans it apparently recruits excision repair proteins to the region of stopped transcription.

Third disease DNA repair is trichotnodystrophy (TCD). With this disease, hypersensitivity to ultraviolet radiation manifests itself in approximately half of the patients.

The disease is accompanied by increased hair fragility, which is associated with a decrease in the concentration of sulfur-containing proteins in them, which have a deficiency of cysteine.

To the main symptom complex include: anomalies in the development of teeth and skin, ichthyosis, delayed sexual development, physical and mental retardation, predisposition to skin cancer.

In a number of cases, it was established that the DNA repair defect in TCD corresponds to the repair defects noted in all PC complementation groups, except for group B.

This correspondence is especially common for the PC gene of group D. In this regard, it was assumed that the PC D gene is polyfunctional and its protein product takes part in excision repair and transcription dependent on RNA polymerase P. In addition, in the cells of patients with TCD they produce not two, but four subunits of the transcription factor TFIIH.

The fourth disease associated with increased photosensitivity and impaired DNA repair is Bloom's syndrome (BS).

The SB gene, or the BLM gene (Bloom mutation), is mapped to the 15q26 locus next to the fes protoncogene. It is assumed that this gene has DNA-dependent ATPase and DNA-dependent helicase activities, the former being associated with the maintenance of chromosome stability in somatic cells, and the latter playing a key role in DNA repair.

Symptoms of SB: proportional pre- and postnatal growth retardation, hyper or hypopigmentation of the skin, redness on

butterfly-shaped face, predisposition to tumors, high level of spontaneous chromosomal aberrations and SCO.

Monogenic diseases associated with double-strand DNA breaks in the complete absence of excision repair

The only example of a disease associated with double-strand DNA breaks in the complete absence of excision repair mechanisms is AT, or Louis-Bar syndrome. The disease occurs with a frequency of 1:40-100 thousand people and is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, high chromosome fragility and a predisposition to malignant tumors (see Chapter 5).

Over the past three decades, a number of genetic features of this disease have been studied. It turned out that the chromosomes of patients with AT have telomeres shortened by almost 3 times; they (chromosomes) are highly sensitive to the action of ionizing radiation and chemical radiomimetics, which is manifested in an increase in the incidence of chromosomal abnormalities (double-strand breaks) and a decrease in the survival of somatic cells, which are characterized by radioresistance DNA synthesis, which is manifested in the absence of synthesis of the p53 protein, which is normally responsible for the delay (stop) of the mitotic cycle at the G 1 -S and G 2 -M stages, necessary for the repair of DNA damage. In other words, cells from patients with AT “simply do not have time” to restore normal DNA structure using excision repair mechanisms. Therefore, post-replicative repair mechanisms are used to eliminate double-strand DNA breaks in such cells.

Monogenic diseases with impaired excision repair not related to photosensitivity and double-strand DNA breaks

Diseases with impaired excision repair not related to photosensitivity and double-strand DNA breaks include: Fanconi anemia (FA) and Hutchinson-Gilford and Werner progeroid syndromes.

Fanconi anemia

AF is a familial hypoor aplastic anemia with congenital deficiency of the hematopoietic sprout of the bone marrow, skin pigmentation disorders, telangiectasia, bipolar disorder and melanoma.

(see Chapter 23), predisposition to myeloid leukemia and other symptoms.

A constant sign of the disease is spontaneous chromosomal instability detected in the cells of the bone marrow, skin and lymphocytes.

FA is a heterogeneous disease that has 8 complementation groups (A, B, C, D, E, F, G and H), including for groups A and C genes mapped to the 9q22.3 locus, but their protein products have not been sufficiently studied . It should be noted that there is inconsistency in the data regarding the absence of increased DNA photosensitivity in patients with FA, although such data were previously obtained (Higurashi M., Cohen P.E., 1975).

Progeria Hutchinson-Gilford

PCP is a rare disease (incidence 1:1 million people). The life expectancy of patients usually does not exceed 15 years. The disease gene is not localized.

Main symptoms: short stature, “bird profile” of the face, predominance of the cerebral part of the skull over the facial part, venous network on the scalp and forehead, dry worn skin, absence of eyebrows and eyelashes, often total alopecia, defects in the number and shape of teeth, complete absence of subcutaneous fat, delayed physical, psychomotor and mental development; in urine - high content of hyaluronic acid. Patients are usually infertile.

Causes of early death: myocardial infarction with generalized atherosclerosis and fibrosis, fatty degeneration of brain tissue and parenchymal organs.

Defects in the repair of DNA-protein cross-links induced by chemical compounds have been established in the cells of patients with PRCH; sharply reduced Hayflick number and its relationship with congenital telomere shortening.

By analogy with KS (see above), an autosomal recessive type of inheritance is assumed for childhood progeria (CP), although a newly emerged autosomal dominant mutation causing telomere shortening is possible.

Werner syndrome

Werner syndrome (WS) or adult progeria is characterized by premature aging, which manifests itself only after puberty. Patients turn gray and bald early (up to 20 years).

The disease gene (WRN gene) is localized in the 8p12-p21 locus, produces the helicase enzyme, but is not part of the main TFIIN transcription complex. It is this feature that distinguishes SV from group B and D PCs and group B SC, in which helicase activity is directly related to transcription-related repair.

Main symptoms:“aging skin” (hyperpigmentation, hyperkeratosis, wrinkling, dryness, telangiectasia), dull voice, changes in internal organs characteristic of an aging body (atherosclerosis of the heart and blood vessels, cataracts, osteoporosis, diabetes mellitus; benign or malignant tumors), in urine - high hyaluronic acid content. The Hayflick number is sharply limited not only in the number of divisions, but also in the duration of the cell cycle (3-5 times lower than normal). Unlike the situation with PRCG, with SV the chromosomes of patients do not have shortened telomeres.

Oncological diseases associated with impaired repair

Since the description of retinoblastoma caused by a deletion of chromosome 13 (13q14; see Chapters 17 and 25), intensive research has begun on the role of gene mutations in hereditary forms of cancer. In many forms of cancer, gene mutations have been shown to impair the repair of mismatches that occur as replication errors (due to small deletions or insertions). Such mutations are similar to the mutation in the mutS gene in E. coli, which leads to defects in mismatch repair.

In humans, a mutation in one copy of the MSN2 gene is highly associated with the development of familial nonpolyposis colon cancer (HNPCC) and endometrial cancer. It has also been established that in these diseases, the second copy of the gene is absent in tumor cells and an extremely high frequency of microsatellite repeats is observed (100 times higher than normal). In addition, forms of breast cancer associated with the BRC1 and BRC2 genes, forms of Alzheimer's disease associated with four genes (PS1-PS4) and the prion protein gene, as well as other examples of gene copying of a number of monogenic and polygenic diseases associated with tumors have been identified in humans (see chapters 17 and 25).

DNA is a linear polymer containing from 70-80 to 10 9 mononucleotides, which are connected by covalent phosphodiester bonds that occur between the pentose hydroxyl group of one nucleotide and the phosphate group of the next nucleotide.

X-ray diffraction analysis data showed that the DNA molecule of most living organisms, with the exception of some phages, consists of two polynucleotide chains, antiparallel directed and oriented in such a way that their sugar-phosphate backbones are on the outside, and their nitrogenous bases are on the inside. The bases are arranged in pairs opposite each other and are connected by hydrogen bonds. Pairing occurs only between complementary (suitable to each other) bases: one purine and one perimedine. The A-T pair is connected by two, and the G-C pair by three hydrogen bonds. The DNA molecule has the shape of a double helix, in which the polynucleotide chains are twisted around an imaginary central axis.

The DNA helix is ​​characterized by a number of parameters:

helix width is about 2 nm;

the pitch or complete turn of the helix is ​​3.4 nm and contains 10 pairs of complementary nucleotides.

DNA has unique properties: the ability to self-duplicate (replication) and the ability to self-heal (repair).

20 proteins: recognizing altered sections of DNA and removing them from the chain, restoring the correct sequence of nucleotides and stitching the restored fragment with the rest of the DNA molecule. 5% of all cellular RNA.

Replication is carried out under the control of a number of enzymes and occurs in several stages. It begins at certain points in the DNA molecule. Special enzymes break the hydrogen bonds between complementary nitrogen bonds, and the helix unwinds. The polynucleotide chains of the parent molecule are kept in an untwisted state and serve as a template for the synthesis of new chains.

With the help of the enzyme DNA polymerase, daughter chains are assembled from the deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP) available in the medium, complementary to the mother chains. Replication occurs simultaneously on both maternal strands, but at different speeds and with some differences. On one of the chains (leading) the assembly of the daughter chain occurs continuously, on the other (lagging) - fragmentarily. Subsequently, the synthesized fragments are mixed using the enzyme DNA ligase. As a result, two DNA molecules are formed from one DNA molecule, each of which has a mother and daughter chains. The synthesized molecules are exact copies of each other and the original DNA molecule. This method of DNA replication is called semi-conservative and ensures accurate reproduction in daughter molecules of the information that was recorded in the mother molecule.

Reparations is the ability of a DNA molecule to “correct” changes that occur in its chains. The restoration of the original DNA molecule involves at least

The listed features of the chemical structure and properties of DNA determine the functions it performs. DNA records, stores, reproduces genetic information, and participates in the processes of its implementation between new generations of cells and organisms.

Ribosomal RNA (rRNA) It is synthesized mainly in the nucleolus, in the region of rRNA genes, and is represented by molecules of various molecular weights that are part of the large or small subunits of ribosomes. rRNA accounts for 85% of the total RNA in a cell.

Transfer RNA (tRNA) makes up about 10% of cellular RNA. There are more than 40 types of tRNA. When implementing genetic information, each tRNA attaches a specific amino acid and transports it to the site of polypeptide assembly. In eukaryotes, tRNAs consist of 70-90 nucleotides and have a cloverleaf-shaped structure.

Ribonucleic acids - RNA - are represented by molecules of various sizes, structures and functions. All RNA molecules are copies of certain sections of the DNA molecule and, in addition to the differences already mentioned, are shorter than it and consist of a single chain. Between individual sections of one RNA chain that are complementary to each other, base pairing (A-U, G-C) and the formation of helical sections are possible. As a result, the molecules acquire a specific conformation.

Matrix, or information, RNA(mRNA, mRNA) synthesized in the nucleus under the control of the enzyme RNA polymerase, complementary to informative DNA sequences, transfers this information to ribosomes, where it becomes a matrix for the synthesis of a protein molecule. Depending on the amount of information copied, the mRNA molecule can have different lengths and makes up about 5% of all cellular RNA.

pairs of others. These changes indeed accompany each cycle of DNA replication, but their frequency is much lower than it should be. This is explained by the fact that most changes of this kind are eliminated due to the action of the repair mechanism (molecular restoration) of the original DNA nucleotide sequence.

The repair mechanism is based on the presence of two complementary chains in the DNA molecule. Distortion of the nucleotide sequence in one of them is detected by specific enzymes. Then the corresponding section is removed and replaced by a new one, synthesized on the second complementary DNA strand. This type of repair is called excision repair, i.e. with “cutting” (Fig. 3.15). It is carried out before the next replication cycle, which is why it is also called

pre-replicative.

Rice. 3.14. Scheme of the correction process during DNA synthesis: I - inclusion in the DNA chain of a nucleotide with an altered (tautomeric) form of cytoeine, which “illegally” pairs with adenine; II - quick transition

cytosine into its normal form disrupts its pairing with adenine; the unpaired 3"-OH end of the synthesized chain prevents its further elongation under the action of DNA polymerase; III - DNA polymerase removes the illegal nucleotide, as a result of which the 3"-OH end paired with the matrix reappears; IV - DNA polymerase continues chain extension at the 3"-OH end

Restoring the original DNA structure requires the participation of a number of enzymes. An important point in triggering the repair mechanism is the detection of an error in the DNA structure. Often such errors occur in the newly synthesized chain during the replication process. Repair enzymes must detect this particular chain. In many species of living organisms, the newly synthesized DNA strand differs from the maternal one in the degree of methylation of its nitrogenous bases, which lags behind synthesis. In this case, the unmethylated chain undergoes repair. DNA strand breaks can also be recognized by repair enzymes. In higher organisms, where DNA synthesis does not occur continuously, but in separate replicons, the newly synthesized DNA strand has breaks, which makes it possible to recognize it.

Restoring the DNA structure when the purine bases of one of its chains are lost involves detecting the defect using the enzyme endonuclease, which breaks the phosphoester bond at the site of damage to the chain. Then the changed section with several adjacent nucleotides is removed by the enzyme exonuclease, and in its place, in accordance with the order of the bases of the complementary chain, the correct nucleotide sequence is formed (Fig. 3.15).

Rice. 3.15. Scheme of excision, pre-replicative DNA repair When one of the bases in the DNA chain changes in the restoration of the original

structures, about 20 DNA glycosylase enzymes take part. They specifically recognize damage caused by deamination, alkylation and other structural transformations of bases. Such modified bases are removed. Areas devoid of bases appear and are repaired, as with the loss of purines. If the normal structure is not restored, for example in the case of deamination of nitrogenous bases, some pairs of complementary bases are replaced by others - the C-G pair can be replaced by a T-A pair, etc. (see section 3.4.2.3).

The formation of thymine dimers (T-T) in polynucleotide chains under the influence of UV rays requires the participation of enzymes that recognize not individual altered bases, but more extensive damage to the DNA structure. The repair process in this case is also associated with the removal of the region carrying the dimer and the restoration of the normal nucleotide sequence by synthesis on the complementary DNA strand.

In the case when the excision repair system does not correct a change that has occurred in one DNA strand, fixation occurs during replication

this change and it becomes the property of both DNA strands. This leads to the replacement of one pair of complementary nucleotides with another or to the appearance of breaks (gaps) in the newly synthesized chain against the changed sections. Restoration of the normal DNA structure can also occur after replication.

Post-replicative repair carried out by recombination (exchange of fragments) between two newly formed double helices of DNA. An example of such post-replicative repair is the restoration of normal DNA structure when thymine dimers (T-T) arise when they are not eliminated spontaneously under the influence of visible light ( light reparation) or during pre-replicative excision repair.

Covalent bonds that arise between adjacent thymine residues make them incapable of binding to complementary nucleotides. As a result, breaks (gaps) appear in the newly synthesized DNA chain, recognized by repair enzymes. Restoration of the integrity of the new polynucleotide chain of one of the daughter DNA is carried out due to recombination with the corresponding normal parent chain of another daughter DNA. The gap formed in the mother chain is then filled by synthesis on a polynucleotide chain complementary to it (Fig. 3.16). A manifestation of such post-replicative repair, carried out by recombination between the chains of two daughter DNA molecules, can be considered the often observed exchange of material between sister chromatids (Fig. 3.17).

Rice. 3.16. Scheme of post-replicative DNA repair: I - the appearance of a thymine dimer in one of the DNA chains;

II - formation of a “gap” in the newly synthesized chain against the changed section of the mother molecule after replication (the arrow shows the subsequent filling of the “gap” with a section from the corresponding chain of the second daughter DNA molecule);

III - restoration of the integrity of the daughter chain of the upper molecule due to recombination and in the lower molecule due to synthesis on the complementary chain

Rice. 3.17. Interchromatid exchanges (indicated by arrows)

During pre-replicative and post-replicative repair, most of the damage to the DNA structure is restored. However, if too much damage occurs in the hereditary material of the cell and some of it is not eliminated, the system of inducible (stimulated) repair enzymes (SOS system) is activated. These enzymes fill the gaps, restoring the integrity of the synthesized polynucleotide chains without strictly observing the principle of complementarity. This is why sometimes the repair processes themselves can serve as a source of permanent changes in the DNA structure (mutations). This reaction also applies to the SOS system.

If in a cell, despite the repair carried out, the amount of damage to the DNA structure remains high, DNA replication processes in it are blocked. Such a cell does not divide, which means it does not pass on the resulting changes to its offspring.

Cell cycle arrest caused by DNA damage, combined with the impossibility of molecular repair of altered hereditary material, can, with the participation of a protein whose synthesis is controlled by the p53 gene, lead to the activation of the process of self-destruction (apoptosis) of the defective cell in order to eliminate it from the body.

Thus, an extensive set of different repair enzymes continuously “inspect” the DNA, removing damaged areas from it and helping to maintain the stability of the hereditary material. The combined action of replication enzymes (DNA polymerase and editing endonuclease) and repair enzymes ensures a fairly low frequency of errors in DNA molecules, which is maintained at a level of 1 × 10-9 pairs of changed nucleotides per genome. Given the human genome size of 3 × 109 nucleotide pairs, this means about 3 errors per replicating genome. At the same time, even this level is sufficient for the formation of significant genetic diversity in the form of gene mutations during the existence of life on Earth.

3.4.2.3. Changes in DNA nucleotide sequences. Gene mutations

Uncorrected changes in the chemical structure of genes, reproduced in successive replication cycles and manifested in the offspring in the form of new variants of traits, are called gene mutations.

Changes in the structure of the DNA that forms a gene can be divided into three groups. Mutations of the first group consist in replacing some bases with others. They account for about 20% of spontaneously occurring gene changes. The second group of mutations is caused by a shift in the reading frame that occurs when the number of nucleotide pairs in the gene changes. Finally, the third group is represented by mutations associated with a change in the order of nucleotide sequences within a gene (inversion).

Mutations by type of replacement of nitrogenous bases. These mutations occur for a number of specific reasons. One of them may be a change in the structure of a base already included in the DNA helix that occurs accidentally or under the influence of specific chemical agents. If such an altered form of the base remains undetected by repair enzymes, then during the next replication cycle it can attach another nucleotide to itself. An example is the deamination of cytosine, which is converted into uracil spontaneously or under the influence of nitrous acid (Fig. 3.18). The resulting uracil, not noticed by the enzymeDNA glycosylase,during replication, it binds to adenine, which subsequently attaches a thymidyl nucleotide. As a result, a couple C-G is replaced in DNA by a pair T-A (Fig. 3.19, I ). Deamination of methylated cytosine converts it to thymine (see Figure 3.18). Thymidyl nucleotide, being a natural component of DNA, is not detected by repair enzymes as a change and, during the next replication, attaches an adenyl nucleotide. As a result, instead of a pair C-G a pair also appears in the DNA molecule T-A (Fig. 3.19, II).

Rice. 3.18. Spontaneous deamination of cytosine

Another reason for base substitution may be the erroneous inclusion in the synthesized DNA chain of a nucleotide carrying a chemically altered form of the base or its analogue. If this error remains undetected by replication and repair enzymes, the changed base is included in the replication process, which often leads to the replacement of one pair with another. An example of this is the addition of a nucleotide with 5-bromouracil (5-BU), similar to thymidyl nucleotide, to the adenine of the mother chain during replication. During subsequent replication, 5-BU more readily attaches guanine rather than adenine. Guanine, during further duplication, forms a complementary pair with cytosine. As a result, the A-T pair is replaced in the DNA molecule by a G-C pair (Fig. 3.20).

Rice. 3. 19. Mutations by type of base substitution (deamination of nitrogenous bases in the DNA chain):

I - conversion of cytosine to uracil, replacement of the C-G pair with a T-A pair;

II - conversion of methyl cytosine to thymine, replacement of the C-G pair with a T-A pair

From the above examples it is clear that changes in the structure of the DNA molecule, such as base replacement, occur either before or during the process of replication, initially in one polynucleotide chain. If such changes are not corrected during repair, then during subsequent replication they become the property of both DNA strands.

Rice. 3.20. Base substitution mutations

(incorporation of a nitrogenous base analogue during DNA replication)

The consequence of replacing one pair of complementary nucleotides with another is the formation of a new triplet in the DNA nucleotide sequence encoding the sequence of amino acids in the peptide chain. This may not affect the structure of the peptide if the new triplet is “synonymous” with the previous one, i.e. will code for the same amino acid. For example, the amino acid valine is encrypted by four triplets: CAA, CAG, CAT, CAC. Replacing the third base in any of these triplets will not change its meaning (degeneracy of the genetic code).

IN In the case when the newly emerged triplet encrypts another amino acid, the structure of the peptide chain and the properties of the corresponding protein change. Depending on the nature and location of the replacement, the specific properties of the protein change to varying degrees. There are cases where the replacement of only one amino acid in a peptide significantly affects the properties of the protein, which manifests itself in changes in more complex characteristics. An example is the change in the properties of human hemoglobin when sickle cell anemia (Fig. 3.21). In such hemoglobin (HbS) (unlike normal HbA) - in the p-globin chains in the sixth position, glutamic acid is replaced by valine. This is a consequence of the replacement of one of the bases in the triplet that encodes glutamic acid (CTT or TTC). The result is a triplet that encrypts valine (CAT or TsAT). In this case, replacing one amino acid in the peptide significantly changes the properties of globin, which is part of hemoglobin (its ability to bind to O2 decreases), and the person develops signs of sickle cell anemia.

IN In some cases, replacing one base with another can lead to the appearance of one of nonsense triplets (ATT, ATC, ACT), which do not encrypt any amino acid. The consequence of such a replacement will be the interruption of the synthesis of the peptide chain. It is estimated that nucleotide substitutions in one triplet lead to the formation of synonymous triplets in 25% of cases; in 2-3 - meaningless triplets, in 70-75% - the occurrence of true gene mutations.

Thus, base substitution mutations can arise either as a result of spontaneous changes in the base structure in one of the strands of an existing DNA double helix, or during replication in a newly synthesized strand. If these changes are not corrected during the repair process (or, conversely, arise during repair), they are fixed in both chains and will then be reproduced in subsequent replication cycles. Therefore, an important source of such mutations is disruption of the replication and repair processes.

Frame shift mutations. This type of mutation accounts for a significant proportion of spontaneous mutations. They occur as a result of the loss or insertion of one or more pairs of complementary nucleotides into the DNA nucleotide sequence. Most of the studied mutations that cause

The repair mechanism is based on the presence of two complementary chains in the DNA molecule. Distortion of the nucleotide sequence in one of them is detected by specific enzymes. Then the corresponding section is removed and replaced by a new one, synthesized on the second complementary DNA strand. This type of repair is called excision repair, i.e. with “cutting” (Fig. 15). It occurs before the next replication cycle, which is why it is also called pre-replicative.

Fig. 14. Scheme of the correction process during DNA synthesis:

I-inclusion in the DNA chain of a nucleotide with an altered (tautomeric) form of cytoeine, which “illegally” pairs with adenine; II - the rapid transition of cytosine to its normal form disrupts its pairing with adenine; the unpaired 3"-OH end of the synthesized chain prevents its further elongation under the action of DNA polymerase; III - DNA polymerase removes the illegal nucleotide, as a result of which the 3"-OH end paired with the matrix reappears; IV - DNA polymerase continues to extend the chain at the 3"-OH end.

Restoring the original DNA structure requires the participation of a number of enzymes. An important point in triggering the repair mechanism is the detection of an error in the DNA structure. Often such errors occur in the newly synthesized chain during the replication process. Repair enzymes must detect this particular chain. In many species of living organisms, the newly synthesized DNA strand differs from the maternal one in the degree of methylation of its nitrogenous bases, which lags behind synthesis. In this case, the unmethylated chain undergoes repair. DNA strand breaks can also be recognized by repair enzymes. In higher organisms, where DNA synthesis does not occur continuously, but in separate replicons, the newly synthesized DNA strand has breaks, which makes it possible to recognize it. Restoring the DNA structure when the purine bases of one of its chains are lost involves detecting the defect using the enzyme endonuclease, which breaks the phosphoester bond at the site of damage to the chain. Then the changed section with several adjacent nucleotides is removed by the enzyme exonuclease, and in its place, in accordance with the order of the bases of the complementary chain, the correct nucleotide sequence is formed (Fig. 15).

Fig. 15. Scheme of excision, pre-replicative DNA repair.

When one of the bases in the DNA chain changes, about 20 DNA glycosylase enzymes take part in restoring the original structure. They specifically recognize damage caused by deamination, alkylation and other structural transformations of bases. Such modified bases are removed. Areas devoid of bases appear and are repaired, as with the loss of purines. If the normal structure is not restored, for example in the case of deamination of nitrogenous bases, some pairs of complementary bases are replaced by others - the C-G pair can be replaced by a T-A pair, etc. .

The formation of thymine dimers (T-T) in polynucleotide chains under the influence of UV rays requires the participation of enzymes that recognize not individual altered bases, but more extensive damage to the DNA structure. The repair process in this case is also associated with the removal of the region carrying the dimer and the restoration of the normal nucleotide sequence by synthesis on the complementary DNA strand.

In the case when the excision repair system does not correct a change that has arisen in one DNA strand, during replication this change is fixed and it becomes the property of both DNA strands. This leads to the replacement of one pair of complementary nucleotides with another or to the appearance of breaks (gaps) in the newly synthesized chain against the changed sections. Restoration of the normal DNA structure can also occur after replication.

There are mechanisms in the cell that can fully or partially restore the original structure of altered DNA. Mutations caused by radiation, chemicals and other factors could theoretically lead to the extinction of a bacterial population if the latter were deprived of the ability to DNA repair. A set of enzymes that catalyze the correction of DNA damage are combined into so-called repair systems, which differ fundamentally in the biochemical mechanisms of “healing” damage. There are three main directions for correcting DNA defects.

1. Direct reversal from damaged DNA to the original structure when changes in DNA corrected by a single enzymatic reaction. For example, removal of an incorrectly attached methyl group at the sixth oxygen atom of guanine using methyltransferase; or cleavage of the thymine dimer resulting from irradiation using photolyase ( recombinational repair).

2. "Cutting out" damage followed by restoration of the original structure (excision repair).

3. Activation of special mechanisms, ensuring survival in case of damage DNA(restoration of the original structure DNA as a result of recombination; correction of erroneous base pairing; translational synthesis on a damaged matrix DNA). These mechanisms do not always lead to complete restoration of the original DNA structure.

Compensation for functions impaired as a result of mutations

Primary mutation can be compensated by a secondary mutation that occurred within the mutated gene (intragenically) or in another gene (extragenically). Changes that eliminate the manifestations of a mutation without correcting the original disorder in DNA, called suppression.

Intragenic suppression caused by a secondary mutation correcting the effects of the primary mutation. For example, a point mutation that results in the synthesis of a defective protein with lost biological activity can be corrected if a secondary point mutation results in encoding an amino acid that retains the protein's configuration and activity. The exact restoration of the original gene structure is called a true reverse mutation ( true reversion). If the effect of the first mutation is compensated by a mutation in another part of the gene, such mutations are called secondary reversions.

Extragenic suppression- suppression of the manifestation of a mutation that occurred in one gene due to a mutation in the second gene.