How does DNA replication occur? Processes of replication and transcription The matrix in the replication process is
Nucleic acids play an important role in ensuring the vital activity of cells of living organisms. An important representative of this group of organic compounds is DNA, which carries all the genetic information and is responsible for the manifestation of the necessary characteristics.
What is replication?
As cells divide, they need to increase the amount of nucleic acids in the nucleus to prevent loss of genetic information during the process. In biology, replication is the duplication of DNA by synthesizing new strands.
The main purpose of this process is to transfer genetic information to daughter cells unchanged without any mutations.
Replication enzymes and proteins
The duplication of a DNA molecule can be compared to any metabolic process in a cell that requires the corresponding proteins. Since replication in biology is an important component of cell division, accordingly, many auxiliary peptides are involved here.
- DNA polymerase is the most important reduplication enzyme, which is responsible for the synthesis of the daughter chain. In the cytoplasm of the cell, during the replication process, the presence of nucleic triphosphates is required, which bring all the nucleic bases.
These bases are monomers of nucleic acid, so the entire chain of the molecule is built from them. DNA polymerase is responsible for the assembly process in the correct order, otherwise the appearance of all kinds of mutations is inevitable.
- Primase is a protein that is responsible for the formation of a primer on the template DNA strand. This primer is also called a primer; it has for the enzyme DNA polymerase the presence of initial monomers, from which further synthesis of the entire polynucleotide chain is possible. This function is performed by the primer and its corresponding enzyme.
- Helicase (helicase) forms a replication fork, which is the divergence of template strands by breaking hydrogen bonds. This makes it easier for polymerases to approach the molecule and begin synthesis.
- Topoisomerase. If you imagine a DNA molecule as a twisted rope, as the polymerase moves along the chain, a positive tension will be formed due to the strong twist. This problem is solved by topoisomerase, an enzyme that briefly breaks the chain and unfolds the entire molecule. After which the damaged area is stitched back together, and the DNA does not experience tension.
- Ssb proteins, like clusters, attach to DNA strands at the replication fork to prevent the re-formation of hydrogen bonds before the end of the reduplication process.
- Ligaza. consists of stitching together Okazaki fragments on the lagging strand of a DNA molecule. This occurs by cutting out the primers and inserting native deoxyribonucleic acid monomers in their place.
In biology, replication is a complex multi-step process that is extremely important during cell division. Therefore, the use of various proteins and enzymes is necessary for efficient and correct synthesis.
Reduplication mechanism
There are 3 theories that explain the process of DNA duplication:
- Conservative states that one daughter nucleic acid molecule is of a template nature, and the second is completely synthesized from scratch.
- Semi-conservative was proposed by Watson and Crick and confirmed in 1957 in experiments on E. Coli. This theory states that both daughter DNA molecules have one old strand and one newly synthesized one.
- The dispersive mechanism is based on the theory that daughter molecules have alternating regions along their entire length, consisting of both old and new monomers.
Now a semi-conservative model has been scientifically proven. What is replication at the molecular level? First, helicase breaks the hydrogen bonds of the DNA molecule, thereby opening both strands to the polymerase enzyme. The latter, after the formation of the seeds, begin the synthesis of new chains in the 5’-3’ direction.
The antiparallel property of DNA is the main reason for the formation of leading and lagging strands. On the leading strand, DNA polymerase moves continuously, and on the lagging strand it forms Okazaki fragments, which in the future will be connected using ligase.
Replication Features
How many DNA molecules are in the nucleus after replication? The process itself involves doubling the cell’s genetic makeup, so during the synthetic period of mitosis, the diploid set has twice as many DNA molecules. This entry is usually marked 2n 4c.
In addition to the biological meaning of replication, scientists have found application of the process in various fields of medicine and science. If in biology replication is the doubling of DNA, then in laboratory conditions the reproduction of nucleic acid molecules is used to create several thousand copies.
This method is called polymerase chain reaction (PCR). The mechanism of this process is similar to replication in vivo; therefore, similar enzymes and buffer systems are used for its occurrence.
conclusions
Replication has important biological significance for living organisms. Transmission during cell division is not complete without doubling DNA molecules, so the coordinated work of enzymes is important at all stages.
1. Initiation.
Replication begins in strictly defined DNA sections - replication origin points - ori (from the English origin - beginning). Here there are specific nucleotide sequences - DNA boxes, recognized by the initiator protein, to which other replication enzymes subsequently bind. Since DNA synthesis occurs only on a single-stranded template, it must be preceded by the obligatory separation of two DNA strands, i.e. preparation of the matrix, which includes the following processes:
· DNA helicases unwind the DNA double helix using the energy of ATP. The site where the strands begin to diverge is called the replication fork because of its characteristic Y-shape.
· DNA topoisomerases relieve topological tension (supercoiling) when unwinding DNA. To do this, the enzyme first breaks the DNA strand, then covalently attaches to the broken end. This bond has significant energy, so the reaction is reversible and does not require additional energy expenditure. Two types of topoisomerases have been discovered: topoisomerase I (introduces single-strand breaks) and topoisomerase II (introduces double-strand breaks in DNA).
· SSB proteins (single-strand DNA-binding proteins) bind to single-stranded regions and stabilize the unbraided duplex, preventing the formation of hairpins.
The DNA template is ready. Now it is necessary to add a complementary chain to each of the chains of the parent DNA molecule from the deoxyribonucleoside triphosphates (dNTPs) present in the cell. Enzymes that catalyze the DNA-matrix-determined addition reaction of deoxyribonucleotides are called DNA polymerases (DNCP).
The first DNA polymerase was discovered in 1957 by A. Kornberg, and in 1959 he was awarded the Nobel Prize for the discovery of the mechanism of DNA biosynthesis.
The most well studied DNAPs in prokaryotes are:
· DNAP I. Functions:
5’-3’ – exonuclease (can remove 5’-terminal nucleotide)
· DNAP II. The role is not entirely clear. Does not participate in replication.
· DNAP III. The main enzyme of replication. Functions:
Polymerase (connects nucleotides with phosphodiester bonds),
3’-5’ – exonuclease (can remove 3’-terminal nucleotide)
DNAPs have two features:
Firstly, DNA polymerases cannot begin DNA synthesis, but can only add new deoxyribonucleotide units to the 3’ end of an existing polynucleotide chain. Therefore, DNAP requires priming. The primer required for DNAP to function consists of RNA (approximately 15-17 nucleotides) and is synthesized by the enzyme primase. Primase binds to helicase and DNA, forming a structure called a primosome. DNAP III then binds to the primer and extends the chain.
Secondly, the synthesis of a new polymerase chain is carried out only in the 5’-3’ direction along the template chain, oriented antiparallel, i.e. 3'-5'. Synthesis of chains in the opposite direction never occurs, so the synthesized chains in the replication fork must grow in opposite directions. The synthesis of one chain (leading, leading) occurs continuously, and the other (lagging) - in fragments. The leading strand grows from the 5' to the 3' end in the direction of the replication fork and requires only one act of initiation. The lagging strand also grows from the 5' to the 3' end, but in the direction opposite to the movement of the replication fork. For the synthesis of the lagging chain, several acts of initiation must occur, resulting in the formation of many short chains (Okazaki fragments), the length of which in prokaryotes is 1000-2000 nucleotides.
At the beginning of each Okazaki fragment there is an RNA primer that must be removed because ribonucleotides should not be present in DNA. DNIP I, due to its 5'-3' exonuclease activity, removes the primer and replaces it with deoxyribonucleotides. The gap between two adjacent Okazaki fragments is closed by the enzyme DNA ligase using the energy of ATP.
2. Elongation (chain lengthening).
A complex of replication enzymes, called the replisome, moves along the DNA template molecule, unwinding it and growing complementary DNA strands.
3. Termination (end of replication).
DNA contains replication termination sites containing specific sequences to which terminator proteins bind, preventing further advancement of the replication fork. DNA synthesis ends.
We previously noted that in addition to polymerase activity, DNAPs also have 3’-5’ exonuclease activity. It is necessary for correction, i.e. removal of an incorrectly inserted nucleotide. DNAP checks the conformity of each nucleotide to the template twice: once before incorporating it into the growing chain, and a second time before incorporating the next nucleotide.
The replication rate in prokaryotes is 500 nucleotides/sec.
Replication methods
· θ-type. The replicative eye expands in opposite directions along the circular DNA molecule. In this case, an intermediate structure is formed, reminiscent of the Greek letter θ. Characteristic of prokaryotes and some viruses.
· σ-type (rolling ring mechanism). Replication begins with the cleavage of a phosphodiester bond in one of the chains of the parent ring molecule. DNAP attaches to the free 3' end and grows a new strand. The intermediate structure has the shape of the letter σ. This type of replication is found in some viruses, in particular in bacteriophage lambda.
· Replication of linear molecules with the formation of several replication forks moving towards each other. Characteristic of all eukaryotes and viruses with linear DNA molecules.
Features of replication in eukaryotes
1. Replication occurs during the S-period of the cell’s mitotic cycle.
2. There are many replicons in one DNA molecule, i.e. There are several origins of replication.
3. DNP polymerases:
· α – DNA polymerase. The main enzyme of replication. It also has primase activity. Synthesizes Okazaki fragments.
· β – DNA polymerase – repair enzyme (removes DNA damage).
· γ – DNA polymerase ensures the synthesis of mitochondrial DNA
· δ – DNA polymerase is involved in the synthesis of the leading strand.
4. The length of Okazaki fragments is 100-200 nucleotides.
5. Replication speed 50 nucleotides/sec.
6. There is an enzyme called telomerase, which extends the 3’ end of DNA before replication, because Each time after replication, the length of the 3' end of a linear DNA molecule decreases by the size of the primer. Disturbances in telomere elongation are associated with carcinogenesis and aging.
Thus, from the material discussed above, we can conclude that the biological meaning of replication lies in the accurate reproduction of genetic information, which is necessary for the hereditary material of daughter cells to be identical to the hereditary material of the mother cell. This is very important both for the development and normal functioning of multicellular organisms and for vegetative reproduction.
10.03.2015 13.10.2015
The preservation of genetic information between generations is ensured by the ability of DNA to duplicate. DNA replication is a complex mechanism for obtaining copies of deoxyribonucleic acid from parent organisms to daughter organisms, which occurs during cell division. In this case, the genetic material encoded in DNA is copied and subsequently divided between new cells. The molecular mechanisms that guarantee accurate replication are now quite well understood and represent a complex process that can be defined in the form of an interactive model.
The process of self-duplication of genes (its replication mechanism) underlies reproduction, preservation of heredity, transmission of its properties to offspring, and the development of an entire multicellular organism from just one fertilized egg. The term replication has another name - DNA reduplication.
Who discovered the replication process?
According to the theory of DNA construction proposed by scientists Watson and Crick in the model of a double helical molecule in 1953, it was finally able to explain how DNA reduplication is carried out - hereditary information is recorded and stored,
Scientists Watson and Crick
and also study the chemical principle of its self-doubling. Strict specificity in base pairing in DNA is determined by the complementarity of nitrogenous bases in both strands, which explains the precision in its synthesis. After all, scientists have shown that during the replication process, a pair of nitrogenous bases, for example, guanine together with cytosine, is stabilized using three hydrogen bonds, and a pair of adenine with thymine is stabilized using two. This is what prevents the incorrect pairing of the bases from which the gene molecule is built.
The hypothesis about a possible replication process was first formulated by the same researchers in 1953. They assumed that from any complementary strand, after separation from another, it is possible to obtain a matrix for synthesizing a new strand. In 1958, the scientist Meselson, together with Stahl, this hypothesis was experimentally confirmed.
According to the theory of Watson and Crick, who proved that DNA replication occurs as follows:
1) breaking of hydrogen bonds with subsequent unwinding of the helical DNA strands;
2) synthesis of new complementary regions on single disconnected parts.
The result is the appearance of two similar genes from a single one, and in any new gene one strand is parental, and the other is newly synthesized. This replication mechanism was called semi-conservative.
Basic principles
The basic principle of self-duplication of genes is easy to imagine if you know the basic structure of double helix genes. According to known data, DNA is presented in the form of a double strand of nucleotides. The genetic information in each of its gene strands is identical, each of which includes a nucleotide sequence that exactly matches the sequence of the second. This identity is possible due to the presence of hydrogen bonds that are directed between two complementary bases from opposite chains. Complementary to each other are G (guanidine) and C (cytosite), as well as A (adenine) and T (thymine). Complementarity is a unique property of DNA. Thanks to it, oppositely directed gene chains are called antiparallel.
Therefore, it is quite simple to imagine that during the self-duplication of molecules, the divergence of strands from double helices is observed, followed by the synthesis of a newly synthesized strand on the original strand according to the principle of connecting complementary nucleotide bases.
DNA replication leads to the emergence of two completely new daughter molecules, double-stranded, but completely indistinguishable from the original molecule. It is worth noting that any of the new molecules includes one mother thread and one newly synthesized one.
DNA polymerase is a catalyst for genome self-duplication
In 1957, scientist A. Kornberg was the first to isolate an enzyme from a bacterium (Escherichia coli), which had unique properties and was able to catalyze replication processes, which was called DNA polymerase. After this, similar substances were found in other organisms.
It was found that this particular enzyme can synthesize new strands on the mother chain from nucleotides. Thanks to these unique properties, the polymerase can sequentially extend a single-stranded gene chain by adding complementary nucleotide bases in a specific direction from its 3′ end.
It is now clear that every cell contains variants of DNA polymerases with different tasks and structures. But their main function is to synthesize exact copies of the original genes.
Doubling mechanism
During the self-duplication of genetic information, scientists identify several main stages that proceed similarly in various forms of living organisms (from bacteria to mammals):
initiation of gene circuits;
unraveling of the double helix;
directly completing the second chain.
1. Initiation of DNA chains. It requires the presence of a pre-synthesized small chain - a seed, only if there is 
which polymerase can add nucleotide bases. In its absence, gene synthesis is impossible. However, in practice it is clear that such primers form enzymes in any organism - DNA primases. This enzyme is not accurate and does not know how to correct the mistakes it makes, so it only serves as the initiator of the synthesis process and is then completely removed from the newly synthesized chain, and its space is completed by the polymerase.
2. Unwinding of the double helical DNA chain. This stage is necessary, because the synthesis of a new DNA chain is possible only on a single-stranded molecule. The place where the double helix splits looks like a fork and is therefore called a replication fork. Here the synthesis of daughter chains by polymerases occurs. It is important to note that such divergence usually begins in certain regions called self-duplication origins and includes about 300 nucleotides.
To open such a stable molecule as DNA, the action of special destabilizing enzymes is necessary - proteins together with DNA helicases, which, when encountering double sections of molecules, make cuts in hydrogen bonds between complementary bases, which separates the chains and thereby advances the replication fork. Protein destabilizers prevent single chains from reconnecting, but make it possible to synthesize new ones.
To make it possible for the replication fork to move along a twisted gene strand and to prevent rotation of a strand that is not yet self-duplicated, regions on the DNA appear that make it possible to unwind. This process occurs with the participation of DNA topoisomerases, which make cuts on the gene strands, allowing the molecules to separate, and subsequently they themselves can repair the resulting damage. They help genes adopt an “undercoiled” form, which has fewer turns, allowing the two strands to more easily separate at the replication fork.
3. Intermittent synthesis stage. Replication processes do not actually occur by continuously adding nucleotides simultaneously on two new strands while moving in both directions. Scientists have shown that the synthesis of daughter chains occurs only in the 3′ direction, which leads to elongation of the 3′ end, and the reading of the template by the polymerase occurs only in the 5′ direction. As it may seem, movement of the replication fork in only one direction is impossible. But that's not true. The solution to this mystery is that synthesis is continuously observed along only one strand of the gene, and along the other, synthesis occurs in segments - 100-1000 nucleotide bases, which are called Okazaki fragments. A thread synthesized continuously is called continuous; a thread synthesized in fragments is called lagging. Researchers have shown that the synthesis of each of these fragments occurs using RNA primers, which are removed from the newly synthesized chain at certain intervals and are completed with nucleotides using the polymerase enzyme.
Cooperative action of enzymes at the replication fork
DNA reduplication occurs with the participation of a complex of enzymes and proteins, capable of rapid movement along genes and capable of coordinatedly carrying out the processes of genetic self-duplication with considerable accuracy. This protein complex can be compared in its efficiency to a “sewing machine”; the “parts” here for making DNA are proteins, and the main sources of energy processes are the hydrolysis reactions of nucleotide bases.
The unwinding of the gene molecule occurs under the influence of DNA helicase, which is complemented by DNA topoisomerase, which is capable of unwinding gene chains, as well as proteins with gene-destabilizing properties that can bind to single strands and prevent them from joining back together.
Directly at the site of the replication fork, polymerases of two chains act. The work of the leading strand polymerase is continuous, and the lagging strand is intermittent when using RNA primers that synthesize DNA primase proteins. Such primases, as well as helicases, form a complex structure called a primosome, which has the ability to move towards the opening of the replication fork and synthesizes RNA primers that stimulate the formation of Okazaki fragments.
The polymerase moves in the same direction. Its movement along the lagging chain is difficult to imagine, but it is explained by scientists - the polymerase superimposes the gene chain, which serves as a template for synthesis, on itself, due to which the polymerase of the lagging chain turns in the opposite direction. This coordination in the movement of polymerases helps ensure coordinated duplication of both strands on which duplication occurs.
The total number of proteins involved in the processes of self-duplication of genes is more than twenty, which makes it possible to carry out this complex, highly ordered, energy-intensive process.
Coordination of mechanisms of genome duplication with cell division
Regardless of whether the cell contains one (prokaryotic organisms) or several chromosomes (eukaryotic organisms) 
organisms) when cells divide, the genome must be completely replicated. The signal for the start of duplication of gene material is the process of binding of the initiator regulatory protein together with the DNA sequence at the origin of replication.
In bacteria, the processes of chromosome reduplication begin at a specific point at which replication processes begin and continue until the doubling of all genetic material is completed. Because bacteria contain a chromosome as their only unit of replication, it has been called a replicon. The beginning of replication processes certainly leads to cell division, which occurs after the complete completion of replication. In this case, each of the genomes passes into a separate daughter cell.
Eukaryotic cells make a complete copy of their chromosomes before dividing. Each chromosome divides into several separate replicons, which are activated gradually as each one replicates once. This allows several independent replication forks to form simultaneously. Replication arrest at a particular fork can occur when it collides with another chromosome, or when the end of a chromosome is reached. Thanks to this, the genetic material of the chromosomes is replicated quite quickly. Each pair of chromosomes then moves to the progeny cells during mitotic division.
Regulation of DNA duplication
DNA replication is considered a key event that occurs during cell division. The fundamental point here is that at the moment of cell division, its DNA has already been replicated. This is achieved through certain mechanisms for regulating replication. There are 3 main stages during DNA replication:
replication initiation stage
elongation process
termination.
The main regulation of the replication process occurs at the initiation stage.
How is regulation carried out? The replication process is not possible from every part of the gene, but only from certain ones, called sites of initiation of the replication process. Depending on the organism, the genome may contain one or more initiation sites. As a rule, initiation sites are located on replicons - special regions that begin replication immediately upon gene synthesis.
Accuracy in DNA duplication
To preserve the genetic material of existing organisms, DNA reduplication must be highly accurate. It is known that the genome of any organism is simply enormous. Scientists have shown that during self-duplication of the mammalian genome, which has a total length of about 3 billion nucleotide bases, a total of 1 to 3 errors are observed. How is such precision achieved? After all, genome synthesis has a significant speed - about 500 nucleotides per second in bacteria and about 50 nucleotides per second in mammals. Today, researchers have identified special error correction mechanisms that help, along with a significant speed of genome synthesis, to obtain its exact copy.
The uniqueness of the correction process lies in two main points. First, during the process of gene synthesis, the polymerase double-checks each inserted nucleotide - the first time before adding it to the synthesized chain. And it checks the second time immediately before inserting the subsequent nucleotide. When an error is found, the synthesis of the gene chain stops until it is corrected. Correction occurs by moving the enzyme in the opposite direction and cutting out the last of the added links, so that the correct - complementary - nucleotide can be inserted into this place. In scientific terms, this means that some of the polymerases also have the ability to perform 3′-terminal hydrolyzing activity, which helps remove nucleotides that are erroneously inserted into new genes.
Interactive replication model
Such a model of DNA replication can be represented as a complex “replication machine” consisting of many complex processes and mechanisms regulated by proteins and enzymes. The interactive model helps to visualize the mechanism that occurs during the replication of genetic material.
This model demonstrates the complementary addition of nitrogenous bases during gene synthesis, which are indicated by conventional symbols of different colors. Moreover, nucleotides are capable of combining only in a certain order (the nucleotide adenine only with thymine, and guanine only with cytosine). The chain synthesis mechanism occurs from left to right. The pentose phosphate backbone of a DNA molecule is symbolically identified by arrows indicating the direction of the 3′ and 5′ ends. A positive result of the reaction is provided by an enzyme - polymerase, which moves along the DNA strand.
As a rule, on an interactive model, DNA replication is clearly displayed when starting the model with the Start button; you can pause the animation with the Stop button, and return the interactive mechanism to its original form with the Reset button.
Replication speed
The speed of replication processes is very high, which allows the synthesis of about 45,000 nucleotides in one minute, while the parental fork rotates at a speed of 4500 revolutions per minute. Due to the possibility of simultaneous replication of genetic information, sometimes in thousands of places at once, in eukaryotic organisms the mechanism of complete doubling of genetic material occurs quite quickly. If this were not possible, then copying the genome would take several months.
The importance of the replication process in genetics
The study of the processes of duplication and preservation of genetic material has always attracted the attention of researchers. Thanks to this, the science of molecular biology arose, which today occupies a special place among other sciences.
In our century, it is in this area of science that discoveries have been made that have made it possible to analyze and decipher the most important processes and mechanisms of one of the main aspects of life - the theory of heredity.
The discoveries made in this area are considered to be the greatest scientific achievements of the 20th century, the significance of which is equal in importance to the discovery of radioactivity.
Research in this area has made it possible to create and develop a number of new biological disciplines - molecular biology, bionics, biocybernetics, which today make it possible to solve a number of problems related to human health, the creation of new varieties of plants and animal species.
DNA replication is the process of doubling parental DNA molecules during the reproduction of cells of living organisms. That is, the replication process precedes cell division. Replication, like transcription and translation, is matrix process. During chain replication, DNA molecules diverge and each of them becomes a template on which a new complementary chain is synthesized. In this case, the nucleotides of the new chains are paired complementary with nucleotides of old chains (A with T, G with C). As a result, two daughter double-stranded DNA molecules are formed, indistinguishable from the parent molecule. Each DNA molecule consists of one strand of the original parent molecule and one newly synthesized strand. This copying mechanism is called semi-conservative. Each newly synthesized chain antiparallel parental. The synthesis of one chain (leading) occurs continuously, and the other (lagging) - pulsed. This mechanism is called semi-continuous.
The structure of the replication fork. Leading thread, lagging thread, Okazaki fragments. see picture.
Key enzymes involved in DNA synthesis.
General structural features of DNA polymerases.
They work on the same principle: they lengthen the DNA chain by adding 1 nucleotide to the 3’ end. The choice is dictated by the requirements of complementarity of the template DNA. Traits:
Several independent domains, cat. together they resemble the right hand of a person. The DNA binds in a small indentation formed by three domains. The basis of the catalytic center is formed by conserved amino acid motifs within the palm domain. The “fingers” correctly position the matrix in the active center. The “thumb” binds DNA at the exit of the enzyme and causes high processivity. In the active center, the most important conserved regions of all three domains are brought together and form a continuous surface. The exonuclease activity is located in an independent domain with its own catalytic site. The N-terminal domain is embedded in the exonuclease domain.
Features of DNA polymerase I.
Participates in the repair of damaged DNA, also plays an auxiliary role in DNA replication - it extends the 3' end of the strand paired with the template strand and allows the gaps to be filled with m/d fragments of lagging strands, extends Okazaki fragments from the 3' ends, while simultaneously removing RNA ribonucleosides seeds, with cat. each Okazaki fragment begins. DNA polymerase I is capable of extending the 3' end of one strand at a break in double-stranded DNA and removing nucleotides from the 5' end of the same break (nick translation) - an important role in the repair system.
DNA polymer" I dominates over all others. This is a 103 kDa polypeptide that can be cleaved into 2 parts: the C-terminal fragment, 68 kDa, Klenow fragment, has polymerase and 3'->5" exonuclease activities; the N-coin fragment, 35 kDa, has 5'- >3" exonuclease activity.
Holoenzyme, DNA polymerase III, replisome.
The holoenzyme is a 900 kDa complex containing 10 proteins, divided into 4 types of subcomplexes:
α, ξ, θ. Contains 2 copies of the catalytic core. α – DNA polymerase activity, ξ – 3’-exonuclease activity, θ – stimulates exonuclease.
Contains 2 subunits τ (tau) - they serve to hold together a minimal enzyme with catalytic activity (α).
2 copies of the clamp – responsible for holding the minimal enzyme on DNA templates. Each consists of a homodimer of β subunits. The main role is to minimize the likelihood of the enzyme separating from the matrix before the copying process is completed.
γ – group of 5 proteins, cat. form a clamp-loader - a device for applying a clamp to the DNA matrix. Consists of 2 δ, 1 γ, 1 ψ and 1 χ subunits.
Replisome is a multienzyme complex in the bacterial replication fork that carries out the process of semi-conservative replication; contains DNA polymerase and a number of other proteins.
Eukaryotic DNA polymerases.
DNA polymerase α - initiates the synthesis of a new strand and a lagging one. Associated with the β-subunit and two small proteins with primase activity, so it can synthesize chains anew. 2 functions: priming and extension = α-primase.
DNA polymerase δ – elongates the leading strand
DNA polymerase ξ – participates in the synthesis of the lagging strand
DNA ligases.
Necessary for connecting DNA chains during replication, repair, and recombination. DNA ligases from E. coli and phage T4 are single peptides capable of joining the ends of two different duplex fragments or broken ends of linear or circular DNA chains. Thus, with the help of DNA ligases, both linear and circular duplex DNA molecules can be formed.
DNA helicases.
Unwinds chains using the energy of ATP hydrolysis. Functions as part of a complex that carries out the movement of the replication fork and the replication of untwisted strands. Several Zelicases can act together to increase speed.
SSB-proteins.
Single-strand binding proteins destabilize the helix, bind to the single-stranded region, thereby stabilizing it, i.e. a section of single-stranded DNA is fixed.
DNA topoisomerasesIAndII, gyrase.
When DNA unwinds, the molecule rotates - a change in secondary and tertiary structures. These processes are catalyzed by a group of enzymes called topoisomerases. They introduce single and double-strand breaks in DNA, which allows the nucleic acid molecule to rotate and become a template. According to the mechanism of action, topoisomerases of the first (I) and second (II) types are distinguished.
Type I topoisomerases (in E. coli - swivelase) - introduce a single-strand break into the DNA molecule, type II topoisomerases (in E. coli - gyrase) - carry out a double-strand break in DNA and transfer DNA strands through the break, followed by cross-linking. At the same time, while performing their functions, topisomerases remain associated with the DNA molecule. In these processes, topoisomerases use a tyrosine residue, which carries out a nucleophilic attack on the phosphate group of DNA to form phosphotyrosine. As a result, the enzymes become covalently bound to the 5' or 3' ends of the DNA at the break site. The formation of such a covalent bond eliminates the need to expend energy when restoring the phosphodiester bond at a single-strand break in the final stages of the reaction. Type I DNA topoisomerases have one catalytic tyrosine residue per monomeric protein molecule, while DNA topoisomerase II dimers contain one catalytic residue per subunit, which creates a stepwise double-strand break in the DNA molecule.
Topoisomerases function as hinges, but their actions are opposite. Topoisomerases I, breaking one of the strands of circular supercoiled DNA, unwind the chains and reduce the number of supercoils. Topoisomerases II convert relaxed, non-supercoiled, closed circular DNA into a supercoil.
Replication stages: initiation, elongation, termination. Initiation of the formation of new DNA chains. Primaza. Primosome. Termination of DNA replication and divergence of daughter helices in prokaryotes.
Termination of replication in linear genomes. The problem of replication of a linear open DNA fragment. Telomeres and telomeric repeats, telomeric loop. Telomerase. The mechanism of telomerase. Features of eukaryotic DNA replication. Replicons of eukaryotes.
As in the case of the biosynthesis of other cellular macromolecules, the replication process is conventionally divided into three main stages: initiation, elongation and termination.
Prokaryotic replication
Initiation
The chromosome of prokaryotes is most often represented a single supercoiled circular molecule with one or two origins of replication. In order for each of the two DNA strands to become a template for the synthesis of a new strand, it is necessary for the DNA strands to straighten out and move away from each other. It has been established that DNA chains do not unwind along their entire length, but over a short section. This is where the replication fork forms, the site of DNA duplication.
When DNA unwinds, the molecule rotates - a change in secondary and tertiary structures. These processes are catalyzed by a group of enzymes called topoisomerases . They introduce single and double-strand breaks in DNA, which allows the nucleic acid molecule to rotate and become a template. According to the mechanism of action, topoisomerases of the first (I) and second (II) types are distinguished.
Initiator proteins “sit” on the untwisted section of the parent DNA molecule, from which replication begins and which is called the origin of replication (or origin, oriC). Initiation of replication in oriC begins with the formation of a complex that includes six proteins DnaA, DnaB, DnaC, HU, gyrase and SSB.
First, proteins bind to the nine-nucleotide sequence DnaA , which form a large aggregate. The origin DNA encircles it, and the DNA strands are separated into a region of three 13-mer sequences. At the next stage, the DnaB (helicase) and DnaC proteins join, forming an aggregate of 480 kDa in size, with a radius of 6 nm. Helicase/ DnaB ensures the rupture of hydrogen bonds between nitrogenous bases in the double strand of DNA, leading to its denaturation, i.e. divergence of threads.
As a result of straightening and denaturation of the DNA double helix, a Y-shaped replication fork is formed (Figure). It is at this replication fork that DNA polymerases synthesize daughter DNA molecules. This piece of DNA looks like a bubble or “eye” in unreplicated DNA. Replication “eyes” are formed in those places where the replication origins are located. When the DNA strands are separated, the molecule becomes quite mobile. All possible violations in the structure of single chains are eliminated due to the action SSB proteins
(single-strand DNA-binding proteins or helix-destabilizing proteins), which, by binding to single strands of DNA, prevent them from sticking together. 
1. When does replication occur?- In the synthetic phase of interphase, long before cell division. The period between replication and prophase of mitosis is called the postsynthetic phase of interphase, during which the cell continues to grow and checks whether duplication has occurred correctly.
2. If there were 46 chromosomes before doubling, how many will there be after doubling?- The number of chromosomes does not change when DNA is doubled. Before duplication, a person has 46 single chromosomes (consisting of one double strand of DNA), and after duplication, 46 double chromosomes (consisting of two identical double strands of DNA connected to each other at the centromere).
3. Why is replication needed?- So that during mitosis, each daughter cell can receive its own copy of DNA. During mitosis, each of the 46 double chromosomes is divided into two single chromosomes; two sets of 46 single chromosomes are obtained; these two sets diverge into two daughter cells.
Three principles of DNA structure
Semi-conservative- each daughter DNA contains one chain from the maternal DNA and one newly synthesized one.
Complementarity- AT/CG. Opposite to adenine of one DNA strand there is always thymine of another DNA strand, and opposite to cytosine there is always guanine.
Antiparallelism- DNA strands lie opposite ends to each other. These ends are not studied in school, so a little more detail (and then into the wilds).
The monomer of DNA is a nucleotide, the central part of the nucleotide is deoxyribose. It has 5 carbon atoms (in the nearest picture, the lower left deoxyribose has numbered atoms). Let's see: a nitrogenous base is attached to the first carbon atom, the phosphoric acid of a given nucleotide is attached to the fifth, the third atom is ready to attach the phosphoric acid of the next nucleotide. Thus, any DNA chain has two ends:
- 5" end, phosphoric acid is located on it;
- The 3" end contains ribose.
The antiparallel rule is that at one end of a double strand of DNA (for example, at the top end of the nearest picture), one strand has a 5" end and the other has a 3" end. It is important for the replication process that DNA polymerase can only extend the 3" end. A DNA chain can only grow at its 3" end.
In this picture, the process of DNA doubling occurs from bottom to top. It can be seen that the left chain grows in the same direction, and the right one grows in the opposite direction.
In the following picture top new chain("leading strand") elongates in the same direction in which duplication occurs. Bottom new chain("lagging strand") cannot extend in the same direction, because there it has a 5" end, which, as we remember, does not grow. Therefore, the lower strand grows with the help of short (100-200 nucleotides) Okazaki fragments, each of which grows in the 3" direction. Each Okazaki fragment grows from the 3" end of the primer ("RNA primers", the primers are red in the figure).
Replication enzymes
Overall direction of replication- the direction in which DNA duplication occurs.
Parental DNA- old (maternal) DNA.
Green cloud next to "Parental DNA"- a helicase enzyme that breaks hydrogen bonds between the nitrogenous bases of the old (mother) DNA chain.
Gray ovals on DNA strands that have just been separated from each other- destabilizing proteins that prevent DNA strands from connecting.
DNA pol III- DNA polymerase, which adds new nucleotides to the 3" end of the upper (leading, continuously synthesized) DNA strand (Leading strand).
Primase- primase enzyme, which makes primer (red Lego piece). Now we count the primers from left to right:
- the first primer is still unfinished, primaza is making it right now;
- from the second primer, DNA polymerase builds DNA - in the direction opposite to the direction of DNA doubling, but in the direction of the 3" end;
- from the third primer the DNA chain has already been built (Lagging strand), she came close to the fourth primer;
- the fourth primer is the shortest because DNA polymerase (DNA pol I) removes it (aka RNA, it has nothing to do with DNA, we only needed the right end from it) and replaces it with DNA;
- The fifth primer is no longer in the picture, it has been completely cut out, leaving a gap in its place. DNA ligase (DNA ligase) stitches this break so that the lower (lagging) DNA strand is intact.
The enzyme topoisomerase is not indicated in the super picture, but it will appear later in the tests, so let’s say a few words about it. Here is a rope consisting of three large strands. If three comrades take hold of these three strands and begin to pull them in three different directions, then very soon the rope will stop unraveling and will curl into tight loops. The same thing could happen with DNA, which is a two-stranded rope, if not for topoisomerase. 
Topoisomerosis cuts one of the two DNA strands, after which (second picture, red arrow) the DNA rotates around one of its strands, so that tight loops are not formed (topological stress is reduced).
Terminal underreplication
From the super picture with replication enzymes, it is clear that in the place left after the removal of the primer, DNA polymerase completes the next Okazaki fragment. (Is it really clear? If anything, the Okazaki fragments in the super-painting are indicated by numbers in circles.) When the replication in the super-painting reaches its logical (left) end, then the last (leftmost) Okazaki fragment will not have a “next”, so there will be no one to complete the DNA on the empty space left after removing the primer.
Here's another drawing for you. The black DNA strand is old, maternal. DNA duplication, unlike the superpattern, occurs from left to right. Since the new (green) DNA on the right has a 5" end, it is lagging and is extended by individual fragments (Okazaki). Each Okazaki fragment grows from the 3" end of its primer (blue rectangle). Primers, as we remember, are removed by DNA polymerase, which at this point completes the next Okazaki fragment (this process is indicated by a red ellipsis). At the end of the chromosome there is no one to fill this section, since there is no next Okazaki fragment, there is already an empty space there (Gap). Thus, after each replication, both 5" ends of the daughter chromosomes are shortened (terminal underreplication).
Stem cells (in the skin, red bone marrow, testes) must divide much more than 60 times. Therefore, the enzyme telomerase functions in them, which lengthens telomeres after each replication. Telomerase extends the overhanging 3" end of the DNA so that it grows to the size of the Okazaki fragment. After this, primase synthesizes a primer on it, and DNA polymerase extends the under-replicated 5" end of the DNA.
Tests
1. Replication is a process in which:
A) transfer RNA synthesis occurs;
B) DNA synthesis (copying) occurs;
C) ribosomes recognize anticodons;
D) peptide bonds are formed.
2. Match the functions of enzymes involved in the replication of prokaryotes with their names.
3. During replication in eukaryotic cells, removal of primers
A) is carried out by an enzyme with only DNAase activity
B) forms Okazaki fragments
B) occurs only in lagging strands
D) occurs only in the nucleus
4. If you extract the DNA of bacteriophage fX174, you will find that it contains 25% A, 33% T, 24% G, and 18% C. How could you explain these results?
A) The results of the experiment are incorrect; there was an error somewhere.
B) One could assume that the percentage of A is approximately equal to that of T, which is also true for C and G. Therefore, Chargaff's rule is not violated, DNA is double-stranded and replicates semi-conservatively.
B) Since the percentages of A and T and, accordingly, C and G are different, DNA is a single strand; it is replicated by a special enzyme that follows a special replication mechanism with a single strand as a template.
D) Since neither A is equal to T and neither G is equal to C, then DNA must be single-stranded; it is replicated by synthesizing the complementary strand and using this double-stranded form as a template.
5. The diagram refers to double-stranded DNA replication. For each of squares I, II, III, select one enzyme that functions in this area.
A) Telomerase
B) DNA topoisomerase
B) DNA polymerase
D) DNA helicase
D) DNA ligase
6. A bacterial culture from a medium with a light nitrogen isotope (N-14) was transferred to a medium containing a heavy isotope (N-15) for a time corresponding to one division, and then returned to a medium with a light nitrogen isotope. Analysis of the DNA composition of bacteria after a period corresponding to two replications showed:
| Options answer |
DNA | ||
| light | average | heavy | |
| A | 3/4 | 1/4 | - |
| B | 1/4 | 3/4 | - |
| IN | - | 1/2 | 1/2 |
| G | 1/2 | 1/2 | - |
7. One rare genetic disease is characterized by immunodeficiency, mental and physical retardation, and microcephaly. Suppose that in a DNA extract from a patient with this syndrome you found almost equal amounts of long and very short stretches of DNA. Which enzyme is most likely missing/defective in this patient?
A) DNA ligase
B) Topoisomerase
B) DNA polymerase
D) Helicase
8. The DNA molecule is a double helix containing four different types of nitrogenous bases. Which of the following statements regarding both replication and the chemical structure of DNA is correct?
A) The base sequences of the two strands are the same.
B) In a double strand of DNA, the content of purines is equal to the content of pyrimidines.
C) Both chains are synthesized in the 5’→3’ direction continuously.
D) The addition of the first base of the newly synthesized nucleic acid is catalyzed by DNA polymerase.
E) The error correction activity of DNA polymerase occurs in the 5’→3’ direction.
9. Most DNA polymerases also have the activity:
A) ligase;
B) endonuclease;
B) 5"-exonuclease;
D) 3"-exonuclease.
10. DNA helicase is a key DNA replication enzyme that unwinds double-stranded DNA into single-stranded DNA. An experiment to determine the properties of this enzyme is described below.
Which of the following statements regarding this experiment is correct?
A) The band appearing at the top of the gel is ssDNA only, 6.3 kb in size.
B) The band appearing at the bottom of the gel is 300bp labeled DNA.
B) If the hybridized DNA is treated with DNA helicase only and the reaction is carried out to completion, the arrangement of the bands looks like that shown in lane 3 in b.
D) If the hybridized DNA is treated with boiling only without helicase treatment, the band arrangement appears as shown in lane 2 in b.
E) If the hybridized DNA is treated with boiled helicase only, the band arrangement looks like that shown in lane 1 in b.
District Olympiad 2001
- All-Russian Olympiad 2001
- International Olympiad 2001
- International Olympiad 1991
- International Olympiad 2008
- District Olympiad 2008
- International Olympiad 2010
The full texts of these Olympiads can be found.
