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1 DNA REPAIR 1 Repair systems 1 Direct repair. Examples 2 Excision repair. Examples and types 3 Repair of DNA replication errors 4 Recombinant (post-replicative) repair in bacteria 5 SOS repair DNA repair systems are quite conserved in evolution from bacteria to humans and are best studied in E. coli. Two types of reparation are known: direct and excisional (from the English. Excision - cutting). Direct repair Direct repair is the simplest way to eliminate damage in DNA, which usually involves specific enzymes that can quickly (usually in one step) eliminate the corresponding damage, restoring the original structure of nucleotides. O6-methylguanine-DNA-methyltransferase 1. This is how, for example, O6-methylguanine-DNA-methyltransferase (suicide enzyme) acts, which removes the methyl group from the nitrogenous base to one of its own cysteine residues. In E. coli, up to 100 molecules of this protein. The protein of higher eukaryotes, similar in function, obviously plays an important role in protection against cancer caused by internal and external alkylating factors. DNA insertase 2. AP sites can be repaired by direct insertion of purines with the participation of enzymes called DNA insertases (from the English insert - insert). photolyase 3. Thymine dimers are “opened” by direct reparation with the participation of photolyases, which carry out the corresponding photochemical transformation. DNA photolyases are a group of light-activated enzymes with a wavelength of nm (visible region), for which they have a special light-sensitive center in their structure. They are widely distributed in nature and found in bacteria, yeast, insects, reptiles, amphibians and humans. These enzymes require a variety of cofactors (FADH, tetrahydrofolic acid, etc.) involved in the photochemical activation of the enzyme. Photolyase E. coli
2 is a protein with a molecular weight of 35 kDa, tightly associated with an oligoribonucleotide with a length of nucleotides necessary for the activity of the enzyme. Examples of direct repair 1. The methylated base O6-mG is dimethylated by the enzyme methyltransferase O6-methylguanine-DNA methyltransferase (a suicide enzyme), which transfers a methyl group to one of its cysteine residues 2. AP sites can be repaired by direct insertion of purines with the participation of enzymes called DNA insertases (from the English insert-insert). SCHEME OF EXAMPLE OF DIRECT REPAIR OF DAMAGES In DNA, the methylated base O6-mG is demethylated by the enzyme methyltransferase, which transfers the methyl group to one of its amino acid residues cysteine. Photolyase DNA attaches to the thymine dimer and, after irradiation with visible light, this dimer is expanded. Excision repair (from English excision - cutting). DEFINITION Excision repair involves the removal of damaged bases from DNA and the subsequent restoration of the normal structure of the molecule.
3 MECHANISM Several enzymes usually take part in excision repair, and the process itself affects not only the damaged nucleotide, but also its adjacent nucleotides. CONDITIONS A second (complementary) strand of DNA is required for excision repair. A general simplified scheme of excision repair is shown in Fig. STAGES The first step in excision repair is the excision of abnormal nitrogenous bases. It is catalyzed by a group of DNA-N-glycosylases - enzymes that cleave the glycosidic bond between deoxyribose and a nitrogenous base. IMPORTANT NOTE: 3 In humans, DNA-N-glycosylases have a high substrate specificity: different enzymes of this family recognize and cut out various abnormal bases (8-oxoguanine, uracil, methylpurines, etc.). In bacteria, DNA-N-glycosylase does not have such substrate specificity. base breaks the sugar-phosphate backbone of the DNA molecule in the AP site sequentially cleaves off several nucleotides from the damaged section of one DNA strand SPECIFIC SEQUENTIAL STEPS OF THIS MECHANISE: As a result of the action of DNA-N-glycosylases, an AP site is formed, which is attacked by the enzyme AP-endonuclease. It breaks the sugar-phosphate backbone of the DNA molecule in the AP site and thereby creates conditions for the work of the next enzyme, exonuclease, which sequentially cleaves off several nucleotides from the damaged section of one DNA strand.
4 WHAT HAPPENS NEXT: 4 In bacterial cells, the vacated space is filled with the appropriate nucleotides with the participation of DNA polymerase I, which targets the second (complementary) DNA strand. Since DNA polymerase I is able to extend the 3'-end of one of the strands at the break in double-stranded DNA and remove nucleotides from the 5'-end of the same break, i.e. carry out nick-translation, this enzyme plays a key role in DNA repair. The final stitching of the repaired sites is carried out by DNA ligase. In eukaryotic (mammalian) cells DNA excision repair in mammalian cells is accompanied by a sharp surge in the activity of yet another enzyme, polyADP-ribose polymerase. In this case, ADP-ribosylation of chromatin proteins (histones and non-histone proteins) occurs, which leads to a weakening of their connection with DNA and opens access to repair enzymes. The donor of ADP-ribose in these reactions is NAD+, whose reserves are severely depleted during excisional repair of damage caused by X-ray irradiation: charges of these proteins and weakening their contact with DNA. WHAT IS A GROUP OF ENZYMES DNA glycosylase cleaves the glycosidic bond between deoxyribose and a nitrogenous base
5 which leads to the excision of abnormal nitrogenous bases 5 DNA glycosylases involved in the elimination of oxidative DNA damage in prokaryotic and eukaryotic cells are very diverse and differ in substrate specificity, spatial structure and methods of interaction with DNA. The most studied DNA glycosylases include: endonuclease III (EndoIII), amido pyrimidine DNA glycosylase (Fpg), Mut T and Mut Y of Escherichia coli. E. coli endonuclease III recognizes and specifically cleaves oxidized pyrimidine bases from DNA. This enzyme is a monomeric globular protein consisting of 211 amino acid residues (molecular weight 23.4 kDa). The gene encoding Endo III has been sequenced and its nucleotide sequence has been established. Endo III is an iron-sulfur protein [(4Fe-4S)2+-protein], which has an element of the supra-secondary structure of the "Greek key" type (helix - hairpin - helix), which serves to bind to DNA. Enzymes with similar substrate specificity and similar amino acid sequences have also been isolated from bovine and human cells. E. coli form amido pyridine-dna-glycosylase "recognizes" and cleaves oxidized heterocyclic bases of the purine series from DNA. EXCISION REPAIR SCHEME STAGE 1 DNA N glycosidase removes the damaged base AP endonuclease introduces a break in DNA EXCISION REPAIR SCHEME 1 DNA N glycosidase removes the damaged base AP endonuclease introduces a break in DNA 2 Exonuclease removes a number of nucleotides
6 3 DNA polymerase fills the vacant site with complementary mononucleotides DNA ligase crosslinks the repaired DNA strand 6 Mut T - a small protein with a molecular weight of 15 kDa, which has nucleoside triphosphatase activity, which preferentially hydrolyzes dgtp to dgmp and pyrophosphate. The biological role of Mut T is to prevent the formation of non-canonical A:G and A:8-oxo-G pairs during replication. Such pairs can appear when the oxidized form of dgtp (8-oxo-dGTP) becomes a substrate for DNA polymerase. Mut T hydrolyses 8-oxo-dGTP 10 times faster than dgtp. This makes 8-oxo-dGTP the most preferred Mut T substrate and explains its functional role. Mut Y is a specific adenine DNA glycosylase that cleaves the N-glycosidic bond between adenine and adenosine deoxyribose, which forms a non-canonical pair with guanine. The functional role of this enzyme is to prevent the T:A-G:A mutation by cleaving an intact adenine residue from the A:8-oxo-G base pair.
7 Nucleotide excision repair (ATP-dependent DNA damage removal mechanism) Recently, special attention has been paid to the ATP-dependent DNA damage removal mechanism in excision repair. This type of excision repair is called nucleotide excision repair (NER). It includes TWO STAGES: 1. removal of oligonucleotide fragments containing damage from DNA and Excinuclease, an enzyme that removes DNA fragments 2. subsequent reconstruction of the DNA chain with the participation of a complex of enzymes (nucleases, DNA polymerase, DNA ligase, etc.). The removal of a DNA fragment occurs on both sides of the damaged nucleotide. The length of the removed oligonucleotide fragments differs between prokaryotes and eukaryotes. Removal of a DNA fragment in prokaryotes So, in E. coli, B. subtilus, Micrococcus luteus, a fragment of nucleotide length is cut out, Removal of a DNA fragment in eukaryotes, and in yeast, amphibians and humans - a fragment consisting of nucleotides. Excinuclease enzyme that removes DNA fragments The cleavage of a DNA fragment is carried out by the enzyme excinuclease (excinuclease). In E. coli, this enzyme consists of 3 different protomers uvra uvr B uvr C, each of which performs a specific function during excisional cleavage of a DNA fragment. These proteins are named after the first letters of the words "ultra violet repair". The uvr A protomer has ATPase activity, binds to DNA in the form of a dimer, carrying out primary damage recognition and binding uvr B The uvr B protomer has: 1. Latent ATPase and latent helicase activity necessary to change conformations and unwind the DNA double helix; 7
8 2. Endonuclease activity, cleaving the internucleotide (phosphodiester) bond from the 3' end of the cleaved fragment. Thus, the protomers uvr A, uvr B, uvr C interact with DNA in a certain sequence, carrying out the ATP-dependent reaction of cleavage of the oligonucleotide fragment from the DNA strand being repaired. The resulting gap in the DNA molecule is repaired with the participation of DNA polymerase I and DNA ligase. The model of excision repair with the participation of the above enzymes is shown in Fig. Excision repairs in humans. Excision repairs in humans are also ATP-dependent and include three main stages: damage recognition, double cutting of the DNA strand, reparative synthesis, and ligation of the repaired strand. However, 25 different polypeptides are involved in human DNA excision repair, 16 of which are involved in the cleavage of the oligonucleotide fragment, being excinuclease protomers, and the remaining 9 carry out the synthesis of the repaired region of the molecule. In the DNA repair system in humans, transcription proteins RNA polymerase II and TFPN, one of the six main eukaryotic transcription factors, play a very significant role. It should be noted that excision repair in prokaryotes, as well as in eukaryotes, depends on the functional state of DNA: transcribed DNA is repaired faster than transcriptionally inactive. This phenomenon is explained by the following factors: chromatin structure, strand homology of transcribed DNA regions, the effect of strand damage and its effect on RNA polymerase. IMPORTANT NOTE: ENCODING DNA CHAIN (information storage chain) DNA MATRIX CHAIN (information is written off from it) 8
9 Major damages such as thymine dimer formation are known to block transcription in both bacteria and humans if they occur on the DNA template strand (damages on the coding strand do not affect the transcription complex). RNA polymerase stops at the site of DNA damage and blocks the work of the transcription complex. 9 Transcription-repair linkage factor (TRCF). In E. coli, enhancement of transcriptional repair is mediated by one special protein, the transcription-repair linkage factor (TRCF). This protein promotes: 1. detachment of RNA polymerase from DNA 2. simultaneously stimulates the formation of a complex of proteins that repair the damaged area. At the end of the repair, RNA polymerase falls into place and transcription continues (see Fig.). So the general scheme of excisional repair 1. DNA-N-glycosylase removes the damaged base 2. AP endonuclease introduces a break in the DNA strand 3. Exonuclease removes a number of nucleotides 4. DNA polymerase fills the vacant site with complementary nucleotides 5. DNA ligase crosslinks the repaired DNA strand Error repair DNA replication by methylation Errors in the pairing of nitrogenous bases during DNA replication occur quite often (in bacteria once per 10 thousand nucleotides), as a result of which nucleotides that are not complementary to the nucleotides of the maternal chain are included in the daughter DNA strand - mismatches (English mismatch does not match). Despite the fact that prokaryotic DNA polymerase I has the ability to self-correct, its efforts to eliminate erroneously attached nucleotides are sometimes insufficient, and then some incorrect (non-complementary) pairs remain in the DNA. In this case, repair occurs using a specific system associated with DNA methylation. The action of this repair system is based on the fact that after replication, after a certain time (several minutes), DNA undergoes methylation. In E. coli, adenine is methylated mainly to form N6-methyl-adenine (N6-mA).
10 Up to this point, the newly synthesized (daughter) strand remains unmethylated. If there are unpaired nucleotides in such a chain, then it undergoes repair: Thus, methylation labels DNA and turns on the replication error correction system. In this repair system, special structures are recognized: the G-N6-mA-T-C sequence and the subsequent deformation in the double helix at the place of lack of complementarity (Fig. below). A rather complex complex of repair enzymes takes part in the elimination of unpaired nucleotides in a hemimethylated DNA molecule, which scans the surface of the DNA molecule, cuts out a section of the daughter chain that resorts to mismatches, and then creates conditions for building it up with the necessary (complementary) nucleotides. Different components of this complex have different nuclease, helicase, and ATPase activities, which are necessary for introducing breaks in DNA and cleavage of nucleotides, unwinding the DNA double helix, and providing energy for the movement of the complex along the repaired part of the molecule. A complex of repair enzymes similar in structure and functions was also found in humans. Recombinant (post-replicative) repair 10 In cases where, for one reason or another, the above repair systems are disrupted, gaps (underrepaired areas) can form in DNA chains, sometimes having very significant sizes, which is fraught with a violation of the replication system and can lead to cell death . In this case, the cell is able to use for the repair of one DNA molecule another DNA molecule obtained after replication, i.e., to involve the recombination mechanism for this purpose. In bacteria In bacteria, the Rec A protein takes part in recombinant repair. It binds to a single-stranded DNA region and involves it in recombination with homologous regions of intact strands of another DNA molecule. As a result, both the broken (containing gaps) and intact strands of the DNA molecule being repaired turn out to be paired with intact strands.
11 complementary DNA regions, which opens up the possibility of repair by the above-described systems. In this case, a certain fragment can be cut out and a gap in the defective chain can be filled with it. The resulting gaps and breaks in the DNA chains are filled with the participation of DNA polymerase I and DNA ligase. SOS-reparation The existence of this system was first postulated by M. Radman in 1974. He also gave the name to this mechanism, including the international distress signal "SOS" (save our souls). Indeed, this system turns on when there is so much damage in the DNA that it threatens the life of the cell. In this case, the activity of a diverse group of genes involved in various cellular processes associated with DNA repair is induced. The inclusion of certain genes, determined by the amount of damage in DNA, leads to cellular responses of different significance (starting with the standard repair of damaged nucleotides and ending with the suppression of cell division). The most studied SOS-repair in E. coli, the main participants of which are the proteins encoded by the Rec A and Lex A genes. coli, and the second one (the Lex A protein) is a transcription repressor of a large group of genes intended for bacterial DNA repair. When it is inhibited or resolved, reparation is activated. Binding of Rec A to Lex A leads to cleavage of the latter and, accordingly, activation of repair genes. In turn, the induction of the bacterial SOS system serves as a danger signal for the lambda phage and leads to the fact that the prophage switches from the passive to the active (lytic) way of existence, thereby causing the death of the host cell. The SOS repair system has been found not only in bacteria, but also in animals and humans. eleven
12 12 Genes involved in SOS repair of DNA damage Genes uvr A, B, C, D Rec A lex A rec N, ruv ssb umu C, D sul A Consequences of gene activation Repair of damage to the secondary structure of DNA Post-replicative repair, induction of the SOS system Switching off the SOS system Repairing double-strand breaks Ensuring recombinational repair Mutagenesis caused by changes in the properties of DNA polymerase Suppression of cell division Conclusion Repair of DNA damage is closely related to other fundamental molecular genetic processes: replication, transcription, and recombination. All these processes are intertwined in common system interactions served by a large number of diverse proteins, many of which are polyfunctional molecules involved in the control of the implementation of genetic information in pro- and eukaryotic cells. At the same time, it is obvious that nature "does not skimp" on control elements, creating the most complex systems for correcting those damages in DNA that are dangerous for the organism and especially for its offspring. On the other hand, in those cases when the reparative capacity is not enough to maintain the genetic status of the organism, there is a need for programmed cell death apoptosis. Molecular Biology M. AKADEMA C.
13 SCHEME OF NUCLEOTIDE EXCISION REPAIR IN E.COLI WITH PARTICIPATION OF EXINUCLEASE 1. TRANSCRIPTIONLY INDEPENDENT MECHANISM 13
14 2. TRANSCRIPTION DEPENDENT MECHANISM 14
15 3. GENERAL STAGE OF REPAIR 15 LEGEND A - uvr protein A B - uvr protein C C - uvr protein C small black triangle sign indicates the location of damage
16 REPAIR SCHEME ASSOCIATED WITH DNA METHYLATION 16
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Lecture outline 1. Types of DNA damage 1. Types of DNA damage 2. DNA repair, types and mechanisms: 2. DNA repair, types and mechanisms: Direct Direct Excisional Excisional Postreplicative Postreplicative SOS repair SOS repair 3. Repair and hereditary diseases 3. Repair and hereditary diseases
The process of restoring the original native DNA structure is called DNA repair, or genetic repair, and the systems involved in it are called repair systems. The process of restoring the original native DNA structure is called DNA repair, or genetic repair, and the systems involved in it are called repair systems. Currently, several mechanisms of genetic repair are known. Some of them are simpler and "switch on" immediately after DNA damage, others require the induction of a large number of enzymes, and their action is extended over time. Currently, several mechanisms of genetic repair are known. Some of them are simpler and "switch on" immediately after DNA damage, others require the induction of a large number of enzymes, and their action is extended over time.
From the standpoint of the molecular mechanism, primary damage in DNA molecules can be eliminated in three ways: From the standpoint of the molecular mechanism, primary damage in DNA molecules can be eliminated in three ways: 1. direct return to the original state; 1.Direct return to original state; 2. cutting out the damaged area and replacing it with a normal one; 2. cutting out the damaged area and replacing it with a normal one; 3. recombination recovery bypassing the damaged area. 3. recombination recovery bypassing the damaged area.
Spontaneous DNA damage Replication errors (appearance of non-complementary base pairs) Replication errors (appearance of non-complementary base pairs) Apurinization (cleavage of nitrogenous bases from a nucleotide) Apurinization (cleavage of nitrogenous bases from a nucleotide) Deamination (cleavage of an amino group) Deamination (cleavage of an amino group)
Induced DNA damage Dimerization (crosslinking of adjacent pyrimidine bases to form a dimer) Dimerization (linkage of adjacent pyrimidine bases to form a dimer) DNA breaks: single and double strand DNA breaks: single and double strands Crosslinks between DNA strands Crosslinks between DNA strands
DIRECT DNA REPAIR This type of repair provides direct restoration of the original DNA structure or removal of damage. This type of repair provides direct restoration of the original DNA structure or removal of damage. A widespread repair system of this kind is the photoreactivation of pyrimidine dimers. A widespread repair system of this kind is the photoreactivation of pyrimidine dimers. This is so far the only known enzymatic reaction in which the activation factor is not chemical energy, but the energy of visible light. This is so far the only known enzymatic reaction in which the activation factor is not chemical energy, but the energy of visible light. This activates the enzyme photolyase, which separates the dimers. This activates the enzyme photolyase, which separates the dimers.
Photorepair Schematically, light repair looks like this: 1. Normal DNA molecule Irradiation with UV light 2. Mutant DNA molecule - formation of pyrimidine dimers. Action of visible light 3. Synthesis of photolyase enzyme 4. Cleavage of dimers of pyrimidine bases 5. Restoration of normal DNA structure
It has been established that most polymerases, in addition to 5"-3"-polymerase activity, have 3"-5"- exonuclease activity, due to which correction is provided possible errors. It has been established that, in addition to 5'-3'-polymerase activity, most polymerases have 3'-5'- exonuclease activity, which ensures the correction of possible errors. This correction is carried out in two stages: first, each nucleotide is checked for compliance with the template before it is included in the growing chain, and then before the next nucleotide is included in the chain. This correction is carried out in two stages: first, each nucleotide is checked for compliance with the template before it is included in the growing chain, and then before the next nucleotide is included in the chain. DNA REPAIR DUE TO EXONUCLASE ACTIVITY OF DNA POLYMERASES
When the wrong nucleotide is inserted, the double helix is deformed. This allows DNA-P to recognize in most cases a defect in the growing chain. If the erroneously inserted nucleotide is unable to form a hydrogen bond with the complementary base, DNA-II will suspend the replication process until the correct nucleotide takes its place. In eukaryotes, DNA-P does not have 3-5 exonuclease activity. When the wrong nucleotide is inserted, the double helix is deformed. This allows DNA-P to recognize in most cases a defect in the growing chain. If the erroneously inserted nucleotide is unable to form a hydrogen bond with the complementary base, DNA-II will suspend the replication process until the correct nucleotide takes its place. In eukaryotes, DNA-P does not have 3-5 exonuclease activity.
Repair of alkylating damage Genetic damage caused by the addition of alkyl or methyl groups can be repaired by removal of these groups by specific enzymes. The specific enzyme O 6 methylguanine transferase recognizes O 6 methylguanine in DNA and removes the methyl group and returns the base to its original form. Genetic damage caused by the addition of alkyl or methyl groups can be repaired by removal of these groups by specific enzymes. The specific enzyme O 6 methylguanine transferase recognizes O 6 methylguanine in DNA and removes the methyl group and returns the base to its original form.
The action of polynucleotide ligase For example, single-strand DNA breaks can occur under the influence of ionizing radiation. The polynucleotide ligase enzyme reconnects the broken ends of DNA. For example, under the influence of ionizing radiation, single-strand breaks in DNA can occur. The polynucleotide ligase enzyme reconnects the broken ends of DNA.
Stages of excision repair 1. Recognition of DNA damage by endonuclease 1. Recognition of DNA damage by endonuclease 2. Incision (cutting) of the DNA strand by the enzyme on both sides of the damage 2. Incision (notching) of the DNA strand by the enzyme on both sides of the damage 3. Excision (cutting and removal ) damage with helicase 3. Excision (cutting and removal) of damage with helicase 4. Resynthesis: DNA-P fills the gap and ligase joins the DNA ends 4. Resynthesis: DNA-P fills the gap and ligase joins the DNA ends
Mismatch repair During DNA replication, mating errors occur when instead of complementary steam A-T, Г-Ц non-complementary pairs are formed. Mismatching only affects the child strand. The mismatch repair system must find the daughter strand and replace non-complementary nucleotides. During DNA replication, mating errors occur when non-complementary pairs are formed instead of complementary pairs A-T, G-C. Mismatching only affects the child strand. The mismatch repair system must find the daughter strand and replace non-complementary nucleotides.
Mismatch repair How to distinguish a child strand from a parent strand? How to distinguish a child chain from a parent chain? It turns out that special methylase enzymes attach methyl groups to adenines in the GATC sequence on the parent chain and it becomes methylated, in contrast to the unmethylated daughter. In E. coli, the products of 4 genes respond to a mismatch repair: mut S, mut L, mut H, mut U. It turns out that special methylase enzymes attach methyl groups to adenines in the GATC sequence on the maternal chain and it becomes methylated, in contrast to unmethylated child. In E. coli, the products of 4 genes correspond to a mismatch repair: mut S, mut L, mut H, mut U.
POST-REPLICATIVE DNA REPAIR Post-replicative DNA repair occurs when the damage survives into the replication phase (too much damage, or the damage occurred immediately before replication) or is of a nature that makes it impossible to repair it with excisional repair (for example, cross-linking of DNA strands). This system plays a particularly important role in eukaryotes, providing the ability to copy even from a damaged matrix (albeit with an increased number of errors). One of the varieties of this type of DNA repair is recombinational repair.
SOS repair Discovered in 1974 by M. Radman. He gave the name by including an international distress signal. Turns on when there is so much damage in the DNA that they threaten the life of the cell. The synthesis of proteins is induced, which attach to the DNA-II complex and build a daughter DNA chain opposite the defective template. As a result, DNA doubles in error and cell division can occur. But if vital functions were affected, the cell will die. Discovered in 1974 by M. Radman. He gave the name by including an international distress signal. Turns on when there is so much damage in the DNA that they threaten the life of the cell. The synthesis of proteins is induced, which attach to the DNA-II complex and build a daughter DNA chain opposite the defective template. As a result, DNA doubles in error and cell division can occur. But if vital functions were affected, the cell will die.
DNA REPAIR AND HUMAN HERITAGE DISEASES Violation of the repair system in humans is the cause of: Premature aging Oncological diseases (80-90% of all cancers) Autoimmune diseases (rheumatoid arthritis, SLE, Alzheimer's disease)
Diseases associated with impaired repair Xeroderma pigmentosa Xeroderma pigmentosum Ataxia-telangiectasia or Louis-Bar syndrome Ataxia-telangiectasia or Louis-Bar syndrome Bloom's syndrome Bloom's syndrome Trichothiodystrophy (TTD) Trichothiodystrophy (TTD) Cockayne's syndrome Cockayne's syndrome Fanconi's anemia Anemia of children (Fanconi's syndrome) Hutchinson-Gilford) Progeria of children (Hutchinson-Gilford syndrome) Progeria of adults (Werner's syndrome) Progeria of adults (Werner's syndrome)
Ataxia-telangiectasia or Louis-Bar syndrome: A-P, cerebellar ataxia, impaired coordination of movements, telangiectases - local excessive expansion of small vessels, immunodeficiency, predisposition to cancer. Bloom's syndrome: A-P, high sensitivity to UV rays, hyperpigmentation, redness on the face in the form of a butterfly.
Trichothiodystrophy: A-P, lack of sulfur in hair cells, brittleness, resembling a tiger's tail, anomalies of the skin, teeth, defects in sexual development. Cockayne syndrome: A-P, dwarfism with normal growth hormones, deafness, optic atrophy, accelerated aging, sensitive to sunlight. Fanconi anemia: a decrease in the number of all cellular elements of the blood, skeletal disorders, microcephaly, deafness. The reason is a violation of the excision of pyrimidine dimers and a violation of the repair of interstrand DNA crosslinks.
Literature: 1. Genetics. Ed. Ivanova V.I. M., Zhimulev I.F. General and molecular genetics. Novosibirsk, Muminov T.A., Kuandykov E.U. Fundamentals of molecular biology (course of lectures). Almaty, Mushkambarov N.N., Kuznetsov S.L. Molecular biology. M., 2003.
DNA REPAIR
Reparation systems
2 Excision repair. Examples and types
3 Repair of DNA replication errors
4 Recombinant (post-replicative) repair in bacteria
5 SOS reparation
DNA repair systems are quite conservative in evolution from bacteria to humans and are best studied in E. coli.
There are two types of reparation:straight and excisional
Direct reparation
Direct repair is the simplest way to eliminate damage in DNA, which usually involves specific enzymes that can quickly (usually in one stage) eliminate the corresponding damage, restoring the original structure of nucleotides.
1. This works, for example,O6-methylguanine-DNA-methyltransferase
(a suicidal enzyme) that removes a methyl group from a nitrogenous base to one of its own cysteine residues
In E. coli, up to 100 molecules of this protein can be synthesized in 1 minute. The protein of higher eukaryotes, similar in function, obviously plays an important role in protection against cancer caused by internal and external alkylating factors.
DNA insertase
2. DNA insertases
photolyase
3. Thymine dimers are "embroidered" by direct reparation starringphotolyase, which carry out the corresponding photochemical transformation. DNA photolyases are a group of light-activated enzymes with a wavelength of 300 - 600 nm (visible region), for which they have a special light-sensitive center in their structure.
They are widely distributed in nature and found in bacteria, yeast, insects, reptiles, amphibians and humans. These enzymes require a variety of cofactors (FADH, tetrahydrofolic acid, etc.) involved in the photochemical activation of the enzyme. E. coli photolyase is a 35 kDa protein tightly bound to oligoribonucleotide 10-15 nucleotides long required for enzyme activity.
Examples of direct reparation
1. Methylated base O 6-mG dimethylated by the enzyme methyltransferaseO6-methylguanine-DNA-methyltransferase (a suicidal enzyme) that transfers a methyl group to one of its residues
cysteine
2. AP sites can be repaired by direct insertion of purines with the help of enzymes calledDNA insertases(from English insert- insert).
SCHEME OF AN EXAMPLE OF DIRECT DAMAGE REPAIR IN DNA - Methylated Base O6- mgdemethylated by the enzyme methyltransferase, which transfers a methyl group to one of its cysteine amino acid residues.
3. Photolyase attaches to the thymine dimer, and after irradiation of this complex with visible light (300-600 nm), the dimer expands
SCHEME OF EXAMPLE OF DIRECT REPAIR OF DAMAGE IN DNA – Photolyase
attaches to the thymine dimer and, after irradiation with visible light, this dimer expands
Excision repair
(from English excision - cutting).
DEFINITION
Excision repair includes removal damaged nitrogenous bases from DNA and subsequent recovery normal molecular structure.
MECHANISM
Several enzymes are usually involved in excision repair, and the process itself affects
not only damaged ,
but also adjacent nucleotides .
CONDITIONS
For excision repair, a second (complementary) strand of DNA is required. The general simplified scheme of excisional repair is shown in fig. 171.
STAGES
The first step in excision repair is excision of the abnormal nitrogenous bases. It's catalyzed by a groupDNA-N-glycosylase- enzymes that cleave the glycosidic bond between deoxyribose and a nitrogenous base.
IMPORTANT NOTE:
AthumanDNA-N-glycosylasehave a high substrate specificity: different enzymes of this family recognize and excise various anomalous bases(8-oxoguanine, uracil, methylpurines, etc.).
AtbacteriaDNA-N-glycosylasedoes not have such substrate specificity.
GENERAL EXCISION REPAIR ENZYMES
| TITLE | FUNCTION | MECHANISM |
| DNA-N-glycosylase | excision of abnormal nitrogenous bases | cleaves the glycosidic bond between deoxyribose and nitrogenous base |
| AP endonuclease | creates conditions for the work of the next enzyme - exonucleases | breaks the sugar-phosphate backbone of the DNA molecule in the AP site |
| exonuclease | cleaves a few nucleotides | sequentially cleaves several nucleotides from the damaged section of one DNA strand |
SPECIFIC SEQUENTIAL STEPS OF THIS MECHANISE:
As a result of action DNA- N-glycosylasean AP site is formed, which is attacked by the enzyme AR-endonuclease. It breaks the sugar-phosphate backbone of the DNA molecule in the AP site and thereby creates the conditions for the work of the next enzyme - exonucleases, which sequentially cleaves off several nucleotides from the damaged section of one DNA strand.
In bacterial cells the vacated space is filled with the corresponding nucleotides with the participation DNA polymerase I oriented to the second (complementary) strand of DNA.
Because DNA polymerase I is able to extend the 3' end of one of the strands at a break in double-stranded DNA and remove nucleotides from the 5' end of the same break,
those. realize “nick-broadcast” , this enzyme plays a key role in DNA repair. The final stitching of the repaired areas is carried out DNA ligase.
In eukaryotic (mammalian) cells
DNA excision repair in mammalian cells is accompanied by a sharp surge in the activity of another enzyme,poly ADP-ribose polymerase . At the same time, it happens ADP-ribosylation of chromatin proteins(histones and non-histone proteins), which leads to a weakening of their connection with DNA and opens access to repair enzymes.
Donor ADP-ribosein these reactionsNAD+, whose reserves are greatly depleted during excisional repair of damage caused by X-ray irradiation:
Negatively charged residues ADP-ribose from the internal composition of the molecule NAD+ join via a radicalglutamine acids or phosphoserineto chromatin proteins, which leads to the neutralization of the positive charges of these proteins and the weakening of their contact with DNA.
WHAT IS A GROUP OF ENZYMES
DNA - glycosylases
cleaves the glycosidic bond between deoxyribose and nitrogenous base
which leads to excision of abnormal nitrogenous bases
DNA - glycosylases , involved in the elimination of oxidative damage to DNA in cells prokaryotes and eukaryotes, are very diverse and differ in substrate specificity, spatial structure, and modes of interaction with DNA.
The most studied DNA glycosylases include:
endonuclease III(EndoIII),
forms amido pyrimidine-DNA-glycosylase (fpg)
Mut T and
Mut Ycoli.
Endonuclease IIIE. coli recognizes and specifically cleaves from DNA oxidized pyrimidine bases.
This enzyme is a monomeric globular protein consisting of 211 amino acids residues (mol. weight 23.4 kDa). The gene encoding Endo III has been sequenced and its nucleotide sequence has been established. Endo III is iron-sulfur protein [(4 Fe-4S )2+-protein], which has the element suprasecondary structure type "Greek key" (spiral - hairpin - spiral), serving to bind to DNA. Enzymes with similar substrate specificity and similar amino acid sequences have also been isolated from bovine and human cells.
Form amido pyridine-DNA-glycosylase E. coli "recognizes" and cleaves oxidized heterocyclic bases of the purine series .
EXCISION REPAIR SCHEME STAGE 1
DNAN
EXCISION REPAIR SCHEME
1 DNANglycosidase removes the damaged base
AR endonuclease breaks DNA
2 Exonuclease removes a number of nucleotides
3 DNA polymerase fills the vacant site with complementary
Mononucleotides
DNA ligase crosslinks the repaired DNA strand
Mut T- a small protein with a molecular weight of 15 kDa, which has nucleoside triphosphatase activity, which is predominantly hydrolyses dGTP to dGMP and pyrophosphate.
Biological role of Mut T is to prevent the formation of non-canonical pairs during replicationA:G and A: 8-oxo-G.
Such pairs can appear when oxidized form
dGTP (8-oxo-dGTP) becomes substrate DNA polymerase.
Mut T hydrolyzes 8-oxo-dGTP10 times faster than dGTP.
It does 8-oxo-dGTPmost preferred substrateMutTand explains its functional role.
Mut Yis a specific adenine-DNA glycosylase that cleaves the N-glycosidic bond between adenine and deoxyribose adenosine, forming a non-canonical pair with guanine.
The functional role of this enzyme is to prevent mutation
T:A - G:A by cleavage of intact residue adeninefrom base pair A: 8-oxo-G.
Nucleotide excision repair
(ATP-dependent DNA damage removal mechanism)
Recently, in excision repair, special attention has been paid to the ATP-dependent mechanism for removing damage from DNA. This type of excision repair is called nucleotide excision repair (NER).
It includes TWO STAGES :
1. removal from DNAoligonucleotide fragments containing damage, and
Excinuclease
2. subsequent reconstruction of the DNA chain with the participation of a complex of enzymes (nucleases, DNA polymerases, DNA ligases, etc.).
The removal of a DNA fragment occurs on both sides of the damaged nucleotide. The length of the removed oligonucleotide fragments differs between prokaryotes and eukaryotes.
Removal of a DNA fragment from prokaryotes
So, in E. coli, B. subtilus, Micrococcus luteus, a fragment of length 12-13 nucleotides
Removal of a DNA fragment from eukaryotes
and in yeast, amphibians and humans, a fragment consisting of 24-32 nucleotides.
Excinuclease- an enzyme that removes DNA fragments
DNA fragment is cleaved by an enzymeexcinuclease(excinuclease). In E. coli, this enzyme consists of 3 different protomers -
uvra
uvr B
uvr C
each of which performs a specific function during excisional cleavage of a DNA fragment. The name of these proteins is given by the first letters of the words"ultra violet repair".
Protomer uvr Ahas ATPase activity, binds to DNA in the form of a dimer, carrying out
primary damage recognition and
bindinguvr B
Protomer uvr B has:
one . Latent ATPase and latent helicase activity necessary to change conformations and unwind the DNA double helix;
2. Endonuclease activity, cleaving the internucleotide (phosphodiester) bond from the sideZ"-endcleaved fragment.
Protomer uvr Cacts like endonuclease, introducing a break in the repaired DNA strand with5"-endcut fragment.
So the protomersuvr A, uvr B, uvr Cinteract with DNA in a certain sequence, carrying out an ATP-dependent reactioncleavage of the oligonucleotide fragment from the DNA strand being repaired.
The resulting gap in the DNA molecule is repaired with the participation of DNA polymerase I and DNA ligase. The model of excisional repair with the participation of the above enzymes is shown in fig. 172.
Excisional repairs in humans
Human excision repairs are also ATP dependent and includethree main stages :
damage recognition,
double cutting of the DNA strand,
reductive synthesis and
ligation of the repaired strand.
However, human DNA excision repair involves
25 different polypeptides ,
16 of which are involved in the cleavage of the oligonucleotide fragment, being protomersexcinucleases,
and the rest 9 carry out the synthesis of the repaired portion of the molecule.
In the DNA repair system in humans, a very significant role is played by transcription proteins -
RNA polymerase II and
TF Monone of the six major transcription factors eukaryote.
It should be noted that excision repair in prokaryotes, as well as in eukaryotes, depends on the functional state of DNA:
transcribed DNA is repaired faster
than transcriptionally inactive.
This phenomenon is explained by the following factors:
chromatin structure,
homology of chains of transcribed DNA regions,
chain damage effect and its effect on RNA polymerase.
IMPORTANT NOTE:
CODING CHAIN DNA (chain of information storage)
MATRIX CHAIN of DNA (information is written off from it)
It is known that such large damages as thymine dimer formation, block transcription in both bacteria and humans if they occur on matrix circuit DNA (damage to coding chains do not affect to the transcription complex). RNA polymerase stops at the site of DNA damage and blocks the work of the transcription complex.
Transcription-repair linkage factor (TRCF) .
In E. coli, the enhancement of transcriptional repair is mediated by one special protein -transcription-repair linkage factor (TRCF) .
This protein contributes :
1. detaching RNA polymerase from DNA
2. simultaneously stimulates the formation of a protein complex,
Carrying out the repair of the damaged area.
At the end of the repair, RNA polymerase falls into place and transcription continues (see Fig.).
So the general scheme of excisional repair
1. DNA-N -glycosylase removes damaged base
2. AR-endonuclease introduces a break in the DNA chain
3. Exonuclease removes a number of nucleotides
4. DNA polymerase fills in the vacated area
Complementary nucleotides
5. DNA ligase cross-links the repaired DNA strand
Repair of DNA replication errors
by methylation
Errors in the pairing of nitrogenous bases during DNA replication occur quite often (in bacteria once per 10 thousand nucleotides), as a result of whichinto the daughter strand of DNA nucleotides that are not complementary to the nucleotides of the maternal chain are included -mismatches(English mismatch n e match).
Despite the fact thatDNA polymerase Ithe prokaryote has the ability to self-correct, its efforts to eliminate erroneously attached nucleotides sometimes not enough, and then some wrong (non-complementary) pairs remain in the DNA.
In this case, reparation occurs using a specific system associated withDNA methylation . The action of this repair system is based on the fact that after replication, after a certain time (several minutes), DNA undergoes methylation.
E. coli methylated primarily adenine with education
N6-methyl-adenine (N6-mA).
Up to this point, the newly synthesized(subsidiary)the chain remains unmethylated.
If there are unpaired nucleotides in such a chain, then it undergoes repair: Thusmethylation marks DNA and
includes error correction system replication.
In this reparation system, special structures are recognized:
subsequenceG-N6-mA-T-Cand next behind it deformation
in the double helix at the site of lack of complementarity (Figure below).
in the elimination of unpaired nucleotides in semi-methylated a rather complex complex of repair enzymes takes part in the DNA molecule, which scans the surface of the DNA molecule,cuts a section of the child chain resorting to mismatcham, and then creates conditions for building up
its desired (complementary) nucleotides.
Different components of this complex have different activities.nuclease,
helicase,
ATPaznoy,
necessary to introduce breaks in DNA and cleave nucleotides, unwind the DNA double helix, and provide energy for the movement of the complex along the repaired part of the molecule.
A complex of repair enzymes similar in structure and functions was also found in humans.
Recombinant (post-replicative) repair
In those cases when, for one reason or another, the above repair systems are disrupted, gaps (underrepaired areas) can form in DNA chains, which have sometimes quite significant, which is fraught with disruption of the replication system and can lead to cell death.
In this case, the cell is able to use for the repair of one DNA molecule another DNA molecule obtained after replication, i.e., to involve for this purpose the mechanismrecombination.
In bacteria
In bacteria, recombinant repair takes partRec A protein. It binds to a single-stranded DNA region and involves it in recombination withhomologous regions of intact strands of another DNA molecule .
As a result, both the broken (containing gaps) and intact chains of the DNA molecule being repaired turn out to be paired with intact complementary DNA regions, which opens up the possibility of repair by the above-described systems.
At the same time, there may be cutting out specific fragment and
fillingwith its help gaps in the defective circuit.
The resulting gaps and breaks in the DNA chains are filled with the participation ofDNA polymerase I and DNA ligase .
SOS reparation
The existence of this system was first postulated by M. Radman in 1974. He also gave the name to this mechanism by including in it the international distress signal "SOS" (save our souls).
Indeed, this system turns on when damage to the DNA becomes so great that it threatens the life of the cell. In this case, the activity of a diverse group of genes involved in various cellular processes associated with DNA repair is induced.
The inclusion of certain genes, determined by the amount of damage in DNA, leads to cellular responses of different significance (starting with the standard reparation of damaged nucleotides and ending suppression cell division).
Most studiedSOS reparationin E. coli, the main participants of which are proteins encoded genes Rec AandLex A.
The first one is a multifunctionalRec A protein participating
in DNA recombination, as well as
in regulation of gene transcription phage lambdathat infects E. coli,
and second (Lex A protein)is an repressor transcription of a large group of genes intended for DNA repair bacteria. When it is inhibited or resolved repair is activated.
Binding Rec A with Lex Aleads to the splitting of the latter and accordingly to activation of repair genes.
In turn, the induction of the bacterial SOS system serves tophage lambda danger signal and causes the prophage to switch from passive to active (lytic) path existence, thus causing host cell death.
The SOS repair system has been found not only in bacteria, but also in animals and humans.
Genes Involved in SOS DNA Damage Repair
| Genes | Consequences of gene activation |
| uvr A, B, C, D | Repair of damage to the secondary structure of DNA |
| Rec A | Post-replicative repair, SOS system inductions |
| lex A | Turning off the SOS system |
| rec N, ruv | Repair of double strand breaks |
| Ensuring recombination repair |
|
| umu C, D | Mutagenesis caused by changes in the properties of DNA polymerase |
| sul A | Suppression of cell division |
Conclusion
Repairing DNA damage is closely related to other fundamental molecular genetic processes: replication, transcription and recombination. All these processes are intertwined into a common system of interactions served by a large number of diverse proteins, many of which are polyfunctional molecules involved in control over the implementation of genetic information in pro- and eukaryotic cells. At the same time, it is clear that nature "don't skimp" on control elements, creating the most complex systems for correcting those damages in DNA that are dangerous for the organism and especially for its offspring. On the other hand, in those cases when the reparative capacity is not enough to maintain the genetic status of the organism, there is a need for programmed cell death -apoptosis..
SCHEME OF NUCLEOTIDE EXCISION REPAIR IN E. COLIWITH THE PARTICIPATION OF EXINUCLEASE
1. TRANSCRIPTIONLY INDEPENDENT MECHANISM
2. TRANSCRIPTION DEPENDENT MECHANISM
3. GENERAL STAGE OF REPAIR
SYMBOLS
A - proteinuvr BUT
B - proteinuvr AT
C - proteinuvr With
small black triangle - the sign indicates the location of damage
REPAIR SCHEME ASSOCIATED WITH DNA METHYLATION
DNA synthesis occurs by a semi-conservative mechanism: each strand of DNA is copied. Synthesis occurs in sections. There is a system that eliminates errors in DNA reduplication (photoreparation, pre-reproductive and post-reproductive repair). The reparation process is very long: up to 20 hours, and complex. Enzymes - restriction enzymes cut out an inappropriate section of DNA and complete it again. Repairs never proceed with 100% efficiency, if it did, evolutionary variability would not exist. The repair mechanism is based on the presence of two complementary chains in the DNA molecule. The distortion of the nucleotide sequence in one of them is detected by specific enzymes. Then the corresponding site is removed and replaced by a new one, synthesized on the second complementary DNA strand. This reparation is called excisional, those. with cutout. It is carried out before the next replication cycle, so it is also called pre-replicative. 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 in the newly synthesized chain against the changed sites. Restoration of the normal DNA structure can also occur after replication. Post-reply reparation is carried out by recombination between two newly formed double strands of DNA. During pre-replicative and post-replicative repair, most of the damaged DNA structure is restored. If in the cell, despite the ongoing repair, the amount of damage remains high, the processes of DNA replication are blocked in it. Such a cell does not divide.
19. Gene, its properties. Genetic code, its properties. Structure and types of RNA. Processing, splicing. The role of RNA in the process of realization of hereditary information.
Gene - a section of a DNA molecule that carries information about the structure of a polypeptide chain or macromolecule. The genes of one chromosome are arranged linearly, forming a linkage group. DNA in the chromosome performs different functions. There are different sequences of genes, there are sequences of genes that control gene expression, replication, etc. There are genes that contain information about the structure of the polypeptide chain, ultimately - structural proteins. Such sequences of nucleotides one gene long are called structural genes. Genes that determine the place, time, duration of the inclusion of structural genes are regulatory genes.
Genes are small in size, although they consist of thousands of base pairs. The presence of a gene is established by the manifestation of the trait of the gene (final product). The general scheme of the structure of the genetic apparatus and its work was proposed in 1961 by Jacob, Monod. They proposed that there is a section of the DNA molecule with a group of structural genes. Adjacent to this group is a 200 bp site, the promoter (the site of adjunction of DNA-dependent RNA polymerase). The operator gene adjoins this site. The name of the whole system is operon. Regulation is carried out by a regulatory gene. As a result, the repressor protein interacts with the operator gene, and the operon begins to work. The substrate interacts with the gene regulators, the operon is blocked. Feedback principle. The expression of the operon is turned on as a whole.
In eukaryotes, gene expression has not been studied. The reason is serious obstacles:
Organization of genetic material in the form of chromosomes
In multicellular organisms, cells are specialized and therefore some of the genes are turned off.
The presence of histone proteins, while prokaryotes have “naked” DNA.
DNA is a macromolecule; it cannot enter the cytoplasm from the nucleus and transmit information. Protein synthesis is possible due to mRNA. In a eukaryotic cell, transcription occurs at a tremendous rate. First, pro-i-RNA or pre-i-RNA appears. This is explained by the fact that in eukaryotes, mRNA is formed as a result of processing (maturation). The gene has a discontinuous structure. The coding regions are exons and the non-coding regions are introns. The gene in eukaryotic organisms has an exon-intron structure. The intron is longer than the exon. In the process of processing, introns are "cut out" - splicing. After the formation of a mature mRNA, after interacting with a special protein, it passes into a system - the informosome, which carries information to the cytoplasm. Now exon-intron systems are well studied (for example, oncogene - P-53). Sometimes the introns of one gene are exons of another, then splicing is not possible. Processing and splicing are able to combine structures that are distant from each other into one gene, so they are of great evolutionary importance. Such processes simplify speciation. Proteins have a block structure. For example, the enzyme is DNA polymerase. It is a continuous polypeptide chain. It consists of its own DNA polymerase and endonuclease, which cleaves the DNA molecule from the end. The enzyme consists of 2 domains that form 2 independent compact particles linked by a polypeptide bridge. There is an intron at the border between two enzyme genes. Once the domains were separate genes, and then they got closer. Violations of such a gene structure leads to gene diseases. Violation of the structure of the intron is phenotypically imperceptible, a violation in the exon sequence leads to mutation (mutation of globin genes).
10-15% of RNA in a cell is transfer RNA. There are complementary regions. There is a special triplet - an anticodon, a triplet that does not have complementary nucleotides - GHC. The interaction of 2 subunits of the ribosome and mRNA leads to initiation. There are 2 sites - pectidyl and aminoacyl. They correspond to amino acids. Synthesis of the polypeptide occurs step by step. Elongation - the process of building a polypeptide chain continues until it reaches a meaningless codon, then termination occurs. The synthesis of the polypeptide ends, which then enters the ER channels. The subunits separate. Different amounts of protein are synthesized in a cell.
DNA repair- this is its repair, i.e., the correction of errors that occur in the structure of the molecule. The word "repair" comes from the English "repair", translated as "repair", "repair", etc.
Errors in the structure of DNA that can be repaired are most often understood as a violation of the sequence of nucleotides - the structural units that make up each strand of DNA. The DNA molecule consists of two strands that are complementary to each other. This means that if damage occurs in one of the circuits, then the second undamaged one can restore the damaged section of the first. In addition, in eukaryotic cells, each chromosome is homologous, that is, containing the same set of genes (but not alleles). In the extreme case, when a site on both strands of the molecule is damaged, it can be copied from the homologous chromosome. Also after the S-phase of the cell cycle, when replication (self-copying) has occurred, each chromosome consists of two double-stranded chromatids identical to each other, i.e., in fact, of two identical DNA molecules. This can also be used to restore the original structure of a damaged molecule.
In the process of evolution, many different cellular molecular mechanisms responsible for DNA repair have emerged. Basically, these are various enzymes and their complexes. Some of them are also involved in replication. Damage to the genes that encode such enzymes is especially dangerous. This leads to the loss of one or another reparation mechanism. In this case, more rapid accumulation of damage and mutations occurs in cells. Often this is the cause of uncontrollably dividing cells, i.e., the appearance of tumors.
On the other hand, if the DNA damage is especially strong, then the mechanism of self-destruction is activated in the cells ( apoptosis). Thus, such cells are not allowed to divide, which means that the next generation will not contain significant DNA damage.
Errors in the DNA structure can occur at various stages of its existence (during synthesis, in pre- and post-synthetic periods), for various reasons (accidentally, under the influence of chemical active substances, radiation, etc.). Also, the changes are different (loss chemical group nucleotide or addition of an additional nucleotide, replacement of a nucleotide with another, establishment of a chemical bond between two adjacent nucleotides, chain break, loss of a site, etc.). Due to this diversity, it is difficult to classify reparation mechanisms. Often they are divided into those that occur during replication, immediately after it, and during the rest of the cell's life cycle. The most studied causes of DNA structure changes and repair methods are listed below.
It should be borne in mind that not all errors are corrected, relatively minor and non-critical ones can be passed on to the next generation of cells and organisms. They can not be called damage, rather - mutations. Most mutations are harmful, but those that are neutral or beneficial under given conditions environment, serve as material for evolution. Thus, the imperfection of DNA repair mechanisms ensured the diversity of life on our planet.
Correction of the nucleotide sequence during replication
DNA polymerases perform the main job of DNA replication by adding nucleotide by nucleotide to a new strand. In addition to the main function, many polymerases are able to remove the last nucleotide incorrectly attached, i.e., not complementary to the nucleotide of the template chain.
The chemical structure of nucleotides can be somewhat modified. At the same time, they begin to connect by hydrogen bonds not with their complementary partners. For example, cytosine must bind to guanine. But its altered form hydrogen bonds to the adenine that thymine was supposed to bind to.
When a new DNA strand is synthesized, the next nucleotide is first hydrogen-bonded to the complementary base of the template. After that, the polymerase binds it to the end of the growing chain with a covalent bond.
However, if it was a modified nucleotide that improperly bound to the complementary base of the parent chain, then it usually quickly reverts to its original form and becomes non-complementary. Hydrogen bonds are broken, and it turns out that the end of the new chain has a freely hanging nucleotide covalently linked to the synthesized chain.
DNA polymerase in this case cannot attach the next nucleotide, and there is nothing left for her but to remove this erroneous nucleotide.
If the hydrogen bonds are not broken, then the chain will continue to grow further behind the wrong nucleotide, and the point mutation will remain. It can be fixed after replication.
Repair immediately after replication
After a new strand of DNA has been synthesized, certain enzyme complexes recognize mismatched bases. At the same time, there is a problem of determining the new and old chains of the DNA molecule. The new one is distinguished by the absence of methylated bases and, in eukaryotes, by the presence of time gaps. According to these features, enzyme complexes identify precisely the newly synthesized chain. Thus, in non-complementary base pairs, the "mistake" is the nucleotide of the new chain.
Once an error is found, other enzymes cut out the entire section of DNA containing the wrong base, not just one nucleotide. After that, the polymerase rebuilds this site, and the ligase sews it with the rest of the chain. This mechanism, when a piece of DNA is cut and re-synthesized, is called excision repair(from the word excision - cutting, cutting), it is quite versatile and is used in many cases of repair, and not only when "checking" DNA immediately after replication.
Repair mechanisms in DNA damage
The DNA of an organism can change not only due to errors during replication. The cell lives, is exposed to adverse external factors, its internal biochemical environment can change, provoking reactions that are detrimental to DNA. As a result, the genetic material is somehow damaged. Depending on the type of damage and its scale, various repair mechanisms are activated, involving slightly different sets of enzymatic complexes.
1. There are enzymes that reverse nucleotide changes in situ without removing DNA segments. In other words, if there was a nucleotide in the chain containing the base guanine (G), which, as a result chemical reaction attached a methyl group and turned into methyl guanine, then the enzyme will turn it back into guanine. Basically, such DNA repair concerns the attachment-detachment of certain groups of atoms.
2. In case of loss of purine bases, excisional repair may occur. In the case of deamination and some other structural changes in bases, glycosylase enzymes cut out only the damaged base of the nucleotide. And only after that the standard excisional repair proceeds.
3. A section is also cut out during the formation of dimers, when two adjacent nucleotides are connected to each other. Typically, these reactions occur as a result of exposure to ultraviolet rays. The formation of a dimer provokes the divergence of complementary strands of DNA in this and nearby areas. A bubble is formed, which is recognized by enzymes. Next, excisional repair starts.
4. There are such severe damage to DNA molecules when the structure of both of its chains is broken in the same place. In this case, according to the principle of complementarity, it is no longer possible to restore one chain from another. One example of such damage may be the breaking of a DNA molecule into two parts, for example, under the action of strong radioactive irradiation.
In the event of damage to both strands of the DNA molecule, recombinative repair can come to the rescue, when a section from a homologous chromosome or sister chromatid is inserted instead of the damaged section. In the event of a break, there are also enzymes that can reattach the broken piece of DNA. However, some of the nucleotides may be lost, which in turn can lead to serious mutations.
Recombinative repair during the presynthetic period of the cell cycle can only occur between homologous chromosomes, since each chromosome during this period consists of only one chromatid. During the post-synthetic period, when chromosomes consist of two identical chromatids, a site can be borrowed from a sister chromatid.
It should be emphasized that the set of alleles in sister chromatids is initially identical (if there was no crossing over). Homologous chromosomes do not. Thus, from the point of view of genetics, real recombination occurs only in the case of an exchange between homologous chromosomes. Although here in both cases we are talking about recombination.
Let's consider such an example. Suppose a thymine dimer has arisen in DNA that has not been repaired prior to replication. In the process of replication, the chains of the original DNA molecule diverge and a new complementary chain is built on each. On the template chain that contains the thymine dimer, a new chain site cannot be built in this region. There is simply no normal pattern here. A gap appears in the daughter strand, while a dimer remains in the mother strand. That is, this DNA molecule "does not know" what the correct nucleotide sequence of the site is.
The only way out in this case is to borrow a piece of DNA from another chromatid. It is carried over from one of her chains. The gap formed here is built up according to the pattern of the complementary chain. The transferred site on the damaged molecule builds up a gap in the daughter chain, while the parent chain will continue to contain a dimer that can be repaired later.