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DNA replication: a tutorial. Regulation of DNA replication Molecular mechanism of replication

Based on the parent DNA molecule. DNA replication is carried out by a complex complex consisting of 15-20 different enzyme proteins called the replisome (English) With the help of special enzymes, the double helix of the parent DNA is unraveled into two strands; on each resulting strand, a second strand is completed, forming two identical daughter DNA molecules, which are then twisted into separate helices. During the subsequent division of the mother cell, each daughter cell receives one copy of a DNA molecule that is identical to the DNA of the original mother cell. This process ensures accurate transfer genetic information from generation to generation.

History of the study

Each DNA molecule consists of one strand of the original parent molecule and one newly synthesized strand. This replication mechanism is called semi-conservative. Currently, this mechanism is considered proven thanks to the experiments of Matthew Meselson and Franklin Stahl (g.). Previously, there were two other models: “conservative” - as a result of replication, one DNA molecule is formed, consisting only of parent chains, and one, consisting only of daughter chains; “dispersive” - all DNA molecules resulting from replication consist of chains, some sections of which are newly synthesized, while others are taken from the parent DNA molecule. The DNA molecule is cut in half and two templates are formed. Two templates emerge from the replication fork. If you imagine them in a straightened form, you can see a ruler of combs that are connected at the ends, but have a gap. Let's imagine that one comb is blue and the other is red. Now we will substitute the lower red one (it is made of five combs, like the upper one) with its fifth end to the third upper one (third upper needle). Let's extend the chain both above and below. How would it turn out: five, three, five, etc. - above and below too. Then two more templates are added to these combs after the templates (combs) leave the replication fork. From one DNA molecule two molecules identical to the parent (if there are no mutations) are obtained; this is called semi-conservatism.

General views

DNA replication is a key event during cell division. It is important that by the time of division the DNA has been replicated completely and only once. This is ensured by certain mechanisms regulating DNA replication. Replication occurs in three stages:

replication initiation
elongation
termination of replication.

Replication regulation occurs mainly at the initiation stage. This is quite easy to implement, because replication can begin not from any DNA section, but from a strictly defined one, called the replication initiation site. There can be either just one or many such sites in the genome. Closely related to the concept of a replication initiation site is the concept replicon . A replicon is a section of DNA that contains an origin of replication and is replicated after DNA synthesis begins from this site. Bacterial genomes, as a rule, represent a single replicon, which means that replication of the entire genome is a consequence of just one act of initiation of replication. Eukaryotic genomes (as well as their individual chromosomes) consist of large number independent replicons, this significantly reduces the total replication time of an individual chromosome. The molecular mechanisms that control the number of replication initiation events at each site during one cell division cycle are called copy number control. In addition to chromosomal DNA, bacterial cells often contain plasmids, which are individual replicons. Plasmids have their own copy number control mechanisms: they can ensure the synthesis of just one copy of the plasmid per cell cycle, or thousands of copies.

Replication begins at the replication initiation site with the unwinding of the DNA double helix, which forms replication fork - site of direct DNA replication. Each site can form one or two replication forks, depending on whether replication is unidirectional or bidirectional. Bidirectional replication is more common. Some time after the start of replication, one can observe in an electron microscope replication eye - a section of a chromosome where DNA has already been replicated, surrounded by longer sections of unreplicated DNA.

At the replication fork, DNA copies a large protein complex (replisome), the key enzyme of which is DNA polymerase. The replication fork moves at a speed of about 100,000 base pairs per minute in prokaryotes and 500-5000 in eukaryotes.

Enzymes and their functions

Enzyme	Function
DNA gyrase	Introduces temporary double-strand breaks in DNA, facilitating its unwinding.
Helicase	Separates the strands of a double-stranded DNA molecule into single strands.
SSB proteins	They bind single-stranded DNA fragments and prevent complementary pairing.
Primaza	Synthesizes an RNA primer (primer) - a short fragment of RNA, which is the initiator in the work of DNA polymerase (the polymerase is not capable of synthesizing DNA from scratch, but can add nucleotides to existing ones).
DNA polymerase	Synthesizes DNA by binding to a primer. It should be noted that the polymerase synthesized one end of the maternal DNA continuously and in one direction, and the second in the opposite direction - in fragments.
Sliding clamp proteins	They surround DNA with a ring and “slide” along it along with the DNA polymerase enzyme moving forward. They prevent the dissociation of the enzyme from the DNA template and increase the efficiency of its work.
RNase H	Removes already unnecessary fragments of RNA primer.
DNA ligase	Stitches together DNA fragments (Okazaki fragments).
Telomerase	Adds special repeating sequences of nucleotides to one end of the DNA chain at telomere sites, thereby compensating for their shortening during division.
Replisome (complex of all replication enzymes)	It moves along the DNA matrix molecule, unwinding it and building up complementary DNA chains.

DNA replication

DNA replication- the process of synthesis of a daughter molecule of deoxyribonucleic acid on the matrix of the parent DNA molecule. During the subsequent division of the mother cell, each daughter cell receives one copy of a DNA molecule that is identical to the DNA of the original mother cell. This process ensures that genetic information is accurately passed on from generation to generation. DNA replication is carried out by a complex enzyme complex consisting of 15-20 different proteins, called English. replisome) .

History of the study

General views

replication initiation
elongation
termination of replication.

Replication regulation occurs mainly at the initiation stage. This is quite easy to implement, because replication can begin not from any DNA section, but from a strictly defined one, called the replication initiation site. There can be either just one or many such sites in the genome. Closely related to the concept of a replication initiation site is the concept replicon . A replicon is a section of DNA that contains an origin of replication and is replicated after DNA synthesis begins from this site. Bacterial genomes, as a rule, represent a single replicon, which means that replication of the entire genome is a consequence of just one act of initiation of replication. Eukaryotic genomes (as well as their individual chromosomes) consist of a large number of independent replicons, which significantly reduces the total replication time of an individual chromosome. The molecular mechanisms that control the number of replication initiation events at each site during one cell division cycle are called copy number control. In addition to chromosomal DNA, bacterial cells often contain plasmids, which are individual replicons. Plasmids have their own copy number control mechanisms: they can ensure the synthesis of just one copy of the plasmid per cell cycle, or thousands of copies.

Molecular mechanism of replication

Enzymes (helicase, topoisomerase) and DNA-binding proteins unwind DNA, keep the template in a diluted state and rotate the DNA molecule. Correct replication is ensured by the exact matching of complementary base pairs and the activity of DNA polymerase, which is able to recognize and correct the error. Replication in eukaryotes is carried out by several different DNA polymerases. Next, the synthesized molecules are twisted according to the principle of supercoiling and further DNA compaction. Synthesis is energy-consuming.

The strands of the DNA molecule diverge, form a replication fork, and each of them becomes a template on which a new complementary strand is synthesized. As a result, two new double-stranded DNA molecules are formed, identical to the parent molecule.

Characteristics of the replication process

Notes

Literature

Preservation of DNA over generations: DNA replication (Favorova O.O., SOZH, 1996)PDF (151 KB)
DNA Replication (animation) (English)

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Replicant (BeOS)
Repnikha

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A detailed consideration of the molecular mechanisms of regulation of DNA replication is beyond the scope of the book, so we will limit ourselves to a few comments on this issue and discuss in more detail only the mechanism of regulation of replication in E. coli, including bacterial plasmids, which is directly related to the functioning of plasmid vectors in bacterial cells .

DNA synthesis is closely related to other processes that prepare cell division, since the transfer of the necessary genetic information from parent cells to daughter cells is vital for descendant cells. The presence of excess genetic information negatively affects cell viability, while its deficiency, resulting from underreplication of DNA, leads to a lethal effect due to the absence of vital genes. However, the process of transferring genetic information from parent cells to daughter cells in eukaryotes is not limited to simple reduplication of chromosome DNA. Thus, insects of many species are characterized by the presence of giant polytene chromosomes that arise as a result of multiple rounds of DNA replication of the original chromatids, not accompanied by their divergence.

Polytenization chromosomes represents a large class of genetic phenomena associated with selective overreplication ( animation) or underreplication of individual genetic loci of eukaryotes. A striking example of this kind is a change in the number of ribosomal RNA genes in animals. Amplification of rRNA genes in amphibian oocytes occurs through the formation of their extrachromosomal (extrachromosomal) copies in the form of circular ribosomal (r) DNA molecules, which are further replicated using the “rolling ring” mechanism. In this case, in each cell only one of hundreds of rDNA repeats is amplified, so that rDNA amplification on one repeat somehow suppresses the amplification process on others, and all the resulting repeats of one oocyte are identical, but differ from the sets of amplified rDNA of other oocytes. Strict stage- and tissue-specificity, as well as selective amplification of only one rDNA repeat indicate the presence of subtle regulatory mechanisms of the replication process in this case as well.

Typical examples of an increase in the number of genes due to their selective replication are Magnification rRNA genes and changes in the number of genes that determine cell resistance to drugs. In the first case, the loss of some rRNA genes in Drosophila as a result of deletion is accompanied by a gradual restoration of their number, while in the second case, in cells under conditions of selective action of a drug that is toxic to them, the number of gene copies necessary for its neutralization increases. In particular, this is characteristic of the dihydrofolate reductase gene in the presence of methotrexate. It is suggested that the change in the copy number of such genes is based on the mechanism of unequal crossing over.

Replication of bacterial chromosomes is closely related to cell metabolism. For example, the frequency of initiation of new rounds of replication depends on the growth rate of bacterial cells, and cells of rapidly growing bacteria may contain chromosomes with several working replication forks, although the replication of one bacterial chromosome requires only two, initiated at a single origin of replication (ori) and diverging in opposite directions. This allows bacteria, under favorable conditions, to spend less time generating than for complete replication of the bacterial chromosome. It is obvious that in order to maintain a strictly ordered nature of replication, there must be subtle mechanisms of regulation of replication at the level of initiation of new rounds. Such mechanisms actually exist.

The most well studied mechanisms at present are the mechanisms of regulation of DNA synthesis in E. coli, including the mechanisms of copy number control in the small plasmid E. coli ColE1, which will be discussed below in more detail due to the importance of these phenomena for genetic engineering.

4.2.1.Initiation of DNA replication in E. coli and its regulation

Replication of chromosomal DNA in bacteria plays a key role in their life cycle. During this process, microorganisms reduplicate their genome, and the resulting daughter genomes are then transferred to daughter cells. The high precision with which bacteria carry out such processes indicates the presence of special mechanisms for their coordination and control.

Structure of the replication start areaoriC. The E. coli chromosome contains a single replication origin region(origin), named oriC, at which replication initiation occurs (Fig. I.47, A). The size of the minimum region of the origin of replication that ensures autonomous replication of the chromosome is 258 bp. (position 11–268 in Fig. I.47). A comparison of the primary structures of the origins of replication of various enterobacteria showed that their sequences are represented by short conservative regions, which are interspersed with divergent DNA segments, the lengths of which, however, are highly conserved. The conserved regions turned out to be binding sites for regulatory proteins separated by spacer sequences. OriC contains five consensus 9-nucleotide DnaA initiator binding sites (non-palindromic repeats), called DnaA boxes. In all enterobacteria, the origins of replication contain 9–14 GATC sites, the positions of eight of which are conserved.

On the left side oriC there is an AT-rich region containing three similar sequences 13 nucleotides long, each starting with GATC. The AT cluster is also localized here, which, together with the left 13-nucleotide sequence, forms the region of the unstable DNA helix ( DNA unwinding element). This section of DNA can be replaced without loss of function with a similar nucleotide composition, but with a different nucleotide sequence.

OriC contains binding sites for proteins that bend DNA, IHF (integration host factor) and FIS (factor for inversion stimulation). Both proteins appear to help the initiator DnaA unwind DNA.

Dimeric protein IciA, consisting of subunits with molecular weight 33 kDa, binds specifically to AT-rich 13-mer repeats. The function of this protein is unknown, as is the function of the Rob protein, which specifically interacts with a 26-nucleotide site on the right side of the DnaA box of R4. DNA near the Rob site exhibits a bend that is more pronounced in molecules fully methylated by Dam methyltransferase (see below). The histone-like protein H-NS interacts with such fully methylated DNA, the binding site of which overlaps with the Rob site. This interaction affects the functioning oriC.

Rice. I.47. Structure of the origin of replication region of the E. coli chromosome ( A) and the scheme for initiating its replication ( b)

HobH is a protein that interacts with one strand of DNA methylated at the origin of replication (hemimethylated origin binding)

Functions of the DnaA protein. The DnaA protein plays a key role in the assembly replisomes– a multicomponent protein complex that carries out bidirectional DNA synthesis. The protein recognizes the origin of replication and attracts the remaining protein components of the replisome to the assembly site.

Stages of initiation of DNA synthesis onoriC . Assembly original complex begins with the interaction of the DnaA protein with the DnaA boxes R1–R4 and M (see Fig. I.47, b). To successfully complete the subsequent stages of replisome assembly, the DnaA protein must be in a complex with ATP and interact with supercoiled oriC. Using an electron microscope, the parent complex is detected as a compact ellipsoidal structure containing 20 DnaA monomers, which covers oriC. The initial complex has a highly ordered structure.

In the presence of ATP in high concentration (5 mM), the initial complex is converted into open complex. In this complex, partial unwinding of AT-rich 13-nucleotide repeats located on the left side occurs oriC. At 37° or higher, the single DnaA protein can mediate DNA unwinding. Formation of the open complex at lower temperatures requires the participation of the structuring protein HU or the host bacterial integration factor IHF. In the open complex, small sections of unwinding DNA are found on the right side oriC between DnaA boxes R2 and R4, which are considered as helicase landing sites.

The DnaB protein is a replication fork helicase and enters an open complex to form prepriming complex I, interacting with single-stranded regions of partially unbraided DNA. Such sites are prepared by the DnaA protein, which displaces the SSB protein from the corresponding sites. DnaB enters prepriming complex I in the form of hexamers that form a complex with six DnaC monomers, each of which binds one ATP molecule. In this complex, the helicase activity of the DnaB protein is blocked. The release of DnaC from the complex occurs as a result of ATP hydrolysis. The consequence of this is the activation of the DnaB helicase and its correct location in the complex. The combination of these events transforms prepriming complex I into prepriming complex II.

Helicase should begin to function at the start of the replication fork on the right side oriC near DnaA boxes R2, R3 and R4. To do this, it must be translocated from the site of its initial entry into the complex to the origin of replication. It is assumed that the translocation is associated with ATP-dependent release of the DnaC protein from the complex, which is accompanied by activation of the helicase.

IN priming complex helicase DnaB interacts with DnaG primase, which plays a key role in ensuring the initiation of replication specifically at oriC. Both of these enzymes ensure the coupling of the functioning of two replication forks moving in opposite directions. In a cell-free system, at low concentrations of primase, replication becomes unidirectional and may not initiate at oriC. In the priming complex, the presence of the DnaA protein is no longer required, and after release from the complex it can be reused to initiate replication on another oriC. It is believed that during the coordinated assembly of two replication forks, a primer is synthesized in one of them, which becomes the primer for the synthesis of the leading strand by another replication fork moving in the opposite direction. Primase in the priming complex functions according to a distributive mechanism. After primer synthesis, it leaves the replication fork and is replaced by a new primase molecule during the formation of the next Okazaki fragment.

During the formation of a replisome, an ATP-dependent formation of a dimeric complex of DNA polymerase III holoenzyme occurs at each replication fork, associated with the 3" ends of the primers (sliding clamp, see above). This is followed by coordinated elongation of primers, accompanied by bidirectional synthesis of leading and lagging DNA strands. In a cell-free system, the starting points for the synthesis of leading strands are localized in oriC near DnaA boxes R2, R3 and R4.

Mechanisms of control of replication initiation in vivo. Initiation of DNA replication in E. coli is regulated at at least three levels: 1) initiation is synchronized with the cell cycle; 2) DNA synthesis in each region of the origin of replication in the cell cycle is initiated only once; 3) initiation occurs synchronously in all regions of the origin of replication present in a given bacterial cell. It has been established that DNA synthesis begins after the mass of a bacterial cell per one region of the origin of replication reaches a certain value called mass of initiation(initiation mass). The DnaA protein is currently considered as the main pacemaker (pacemaker), playing a key role in controlling the initiation of replication.

Suppression of protein synthesis in vivo is accompanied by the completion of already initiated DNA synthesis against the background of the cessation of new rounds of initiation. Resumption of protein synthesis leads to the initiation of replication after a lag period of one cell generation. In the presence of all the necessary proteins, initiation is sensitive to rifampin, a specific inhibitor of bacterial RNA polymerase, which indicates the dependence of initiation on the synthesis of untranslated RNA.

The role of topologyoriCin replication initiation. Topoisomerase I and topoisomerase II (DNA gyrase) maintain the bacterial chromosome in a negatively supercoiled state. Approximately half of the supercoiling is neutralized by the histone-like proteins HU, IHF, and FIS, while the remaining supercoiling of the bacterial chromosome facilitates transcription, replication, and site-specific recombination. The bacterial chromosome is thought to consist of 40–50 supercoiled domains with ∼25 supercoils per kb. DNA. There is currently no precise data on the topological state oriC, required for the initiation of replication in E. coli. It is known that mutations in the topoisomerase gene topA suppress temperature-sensitive mutations dnaA(Ts). It is assumed that in these mutant strains the topology oriC modified in such a way that it allows initiation of replication at lower intracellular concentrations of the DnaA protein. In addition, the importance of a particular topological state oriC for initiation indicates the fact that initiation is disrupted in mutant bacteria with an altered gene gyrB(Ts), encoding the B subunit of DNA gyrase.

Activation of replication by transcription. In the event that supercoiling of minichromosomes or plasmids containing oriC, is not sufficient to initiate their replication; initiation can occur with simultaneous transcription of DNA in the vicinity oriC. Changing the topology oriC in this case can be achieved through education R-loops(DNA–RNA hybrid in double-stranded DNA) or due to transcription as such, in which local positive supercoiling of DNA takes place before the transcribing RNA polymerase, followed by negative supercoiling. This facilitates the formation of open complexes during the initiation of DNA synthesis.

The role of the DnaA protein in the regulation of replication initiation. It takes ~60 minutes for bacteria to replicate chromosomal DNA, separate daughter chromosomes, and prepare for a new division. Consequently, cells with a generation time shorter than this period (for example, at elevated temperatures in rich nutrient media) must initiate replication of chromosomes destined for subsequent divisions before completion of the previous round of replication. Thus, a single cell may contain a replicating chromosome with multiple origins of replication. In this case, the initiation of replication at multiple origins of replication occurs simultaneously.

Overproduction of DnaA in bacteria leads to a sharp increase in the frequency of replication initiations without changing the overall rate of DNA synthesis, which indicates DnaA as a positive regulator of this process. Among the models explaining the mechanism of the regulatory action of the DnaA protein, the DnaA titration model is the most widely used. According to this model, all newly synthesized DnaA protein is bound (titrated) by DnaA boxes oriC chromosomes. As soon as the number of initiator molecules exceeds the number of intracellular DnaA boxes (all DnaA boxes are occupied by the protein), DNA synthesis is initiated. After starting initiation on one oriC there is a release of DnaA molecules, a sharp increase in its intracellular concentration and synchronous initiation of DNA synthesis at other accessible regions of the origin of replication. Moreover, the association with membranes is the first oriC protects it from being used in reinitiation.

The role of Dam methylation in the initiation of DNA synthesis. As mentioned above, E. coli Dam methyltransferase modifies adenine residues in 5"-GATC sequences. As a result of replication, the DNA molecule temporarily transforms from a fully methylated molecule to a single-strand methylated one, which allows the cell to recognize newly synthesized DNA. The location of Dam clusters is sites in oriC enterobacteria are highly conservative (see Fig. I.47, A). Unmethylated or half-methylated plasmid DNA does not replicate in dam mutant cells, although it serves as a substrate in the cell-free replication system. Replication of chromosomal DNA in dam mutants begins at oriC, however, replication control is broken, which manifests itself in asynchronous replication on multiple oriC. It turned out that only half methylated, but not fully methylated or unmethylated oriC- DNA binds specifically to the membrane fraction of E. coli in vitro. Moreover, in rapidly growing cells 1/3 of the generation time oriC- DNA is in a half-methylated state, after which it becomes fully methylated. The same is true for the promoter of the initiator gene DnaA, in which the half-methylated state is associated with suppression of gene transcription. In contrast, remethylation of the newly synthesized DNA strand of the rest of the bacterial chromosome occurs quickly—within 1–2 minutes. Based on this kind of data, it is suggested that in an incompletely methylated state, the above-mentioned sequences are shielded by bacterial membranes from contacts with regulatory proteins and cannot participate in the second round of replication initiation (period eclipse). Mutations in the gene seqA sharply reduce the eclipse time, which is manifested in asynchrony of replication initiations. The SeqA protein turned out to be a negative regulator of replication initiation, acting at the interaction stage oriC with bacterial membranes.

The role of the SeqA protein in the regulation of bacterial chromosome replication. Gene seqA encodes a protein of 181 amino acid residues, the inactivation of which is lethal to bacterial cells. A study of the interaction of this protein with unmethylated, partially and fully methylated regions of the origin of replication using the band shift method in polyacrylamide gel electrophoresis showed its preferential binding to partially methylated sequences. However, for full (context-dependent) specificity of its interaction, the presence of additional factors is required. Indeed, as part of DNA-protein complexes formed with the participation of partially methylated sequences oriC, a protein with a molecular weight of 24 kDa was discovered that specifically interacts with the methylated DNA strand in oriC. Screening of the E. coli sequence library allowed the gene to be cloned hobH(hemimethylated origin binding), encoding this protein. Mutations in this gene led to a partial loss of synchronization in replication initiations in bacterial cells, which also indirectly indicates the participation of the HobH protein in the regulation of replication initiation of bacterial chromosomes at the early stages of the cell cycle. However, the true role of this protein in replication is not completely known.

The eclipse period may end as a result of the gradual completion of methylation of a partially methylated sequence oriC, located in complex with membranes. Complete methylation of these sequences prevents their interaction with membranes and makes them accessible to the initiator DnaA.

Termination of replication. The meeting of two replication forks at the end of the replication cycle of a bacterial chromosome is accompanied by several events that are necessary for the complete separation of the two resulting bacterial chromosomes before cell division. The movement of replication forks towards each other is accompanied by homologous recombination between daughter chromatids. If the number of recombinations that have occurred is odd, a bacterial chromosome dimer is formed, while if the number of recombinations is even, two catenated (linked to each other) chromosomes are formed. In the second case, the separation of catenanes by topoisomerase IV leads to complete separation of the daughter chromosomes, whereas in the case of a bacterial chromosome dimer this is not enough. Dimer separation to form monomers occurs as a result of site-specific recombination at the locus dif under the action of resolvase (site-specific recombinase) XerCD.

4.2.2.Regulation of ColE1 plasmid replication

Many prokaryotic cells contain, in addition to the main chromosome, small extrachromosomal DNA called plasmids. Plasmids, whose sizes vary from several thousand to hundreds of thousands of base pairs, and the number of copies per cell from one to several hundred, are capable of autonomous (independent of the main chromosome) replication and are stably inherited over a number of cell generations. Although many plasmids provide significant selective advantages to host cells (resistance to antibiotics, heavy metals, etc.), most of them are cryptic, i.e. not manifested in a visible cell phenotype. Since their existence places a significant burden on the metabolism of host cells, the meaning of their evolutionary stability remains unclear. Although, under natural conditions, bacterial cells do not appear to experience selection pressure to retain plasmids within cells, the latter, through subtle mechanisms that regulate the number of their copies in cells, stably segregate between daughter bacterial cells.

The origin of replication of the small plasmid ColE1, which carries colicin resistance genes, is traditionally used in genetic engineering in the construction of vector DNA molecules, which are used for cloning and expression of short nucleotide sequences in E. coli cells. That is why it is advisable to consider the mechanisms of control of replication of the ColE1 plasmid.

Initiation of ColE1 plasmid replication. Replication of the ColE1 plasmid occurs in one direction (unidirectional replication) using the host cell replication apparatus. The plasmid itself does not encode any enzyme that would be required for its replication. The origin of replication region contains two promoters, one of which provides the synthesis of the RNA primer (RNA II) necessary for the initiation of plasmid replication. The synthesized RNA II, the length of which depends on the type of plasmid being replicated, is further processed by RNase H to produce an RNA of 550 nucleotides in length. This molecule is effectively used by DNA polymerase I as a primer in the synthesis of the leading strand of DNA. In the absence of RNase H, the 3' end of RNA II serves as a primer during replication, although with less efficiency. In cells deficient in RNase H and DNA polymerase, the initiation of ColE1 replication is carried out by DNA polymerase III with the participation of RNA II according to the mechanism discussed in detail above.

All three mechanisms of initiation of plasmid replication are based on the unique property of RNA II to form a stable DNA–RNA hybrid at the origin of replication. Indeed, regular transcripts are released from the transcription complex after completion of transcription and separation of RNA polymerase from the template, which does not happen with RNA II. Analysis of plasmid mutants defective in replication, as well as their revertants, showed that in a stable hybrid of RNA II with the template, interaction occurs between the G-rich loop of RNA II, formed 265 nucleotides upstream of the replication initiation point (position –265), and the C-rich region DNA located in the vicinity of nucleotide –20 (Fig. I.48, A). Both of these sequences were found to be conserved among the related plasmids pMB1, p15A, and KSF1030. Interactions between these sequences apparently occur at a time when the RNA polymerase is still in the transcription complex and the DNA chains in the vicinity of the complex are unwoven. The equilibrium between the two alternative conformations of RNA II is critical in determining the proportion of RNA molecules remaining in the DNA–RNA hybrid required to initiate plasmid replication. The choice between two alternative conformations of RNA II is determined by the primary structure of the region located between nucleotides –359 and –380 (sequence ) (see Fig. I.48, b). This sequence can interact with an upstream complementary sequence  (structure ) or with a homologous sequence  located below (structure ). After RNA polymerase transcribes the first 200 nucleotides, the resulting RNA II forms a temporary secondary structure characterized by three stem-loop domains (I, II, and III). Extension of RNA II by a few more nucleotides leads to the destruction of stem III and the formation of stem IV, which is stabilized as a result of complementary interactions between the  and  sequences. During subsequent elongation of RNA II, it has two alternative opportunities to form its secondary structure. The choice in favor of one conformation or another depends on whether the  sequence remains associated with the  sequence or forms new contacts with the -sequence. The transition from complementary pairs  to  is accompanied by strong changes conformations of RNA II, which ultimately determine its ability to serve as a primer during plasmid replication. RNA II molecules in the  conformation can form an RNA–DNA hybrid, which serves as a substrate for RNase H, but in the  conformation they do not have this ability. The proposed model is confirmed, first of all, by the fact that mutations that favor the formation of the  conformation due to destabilization of stem IV impede the functioning of RNA II as a primer and lead to a decrease in the number of copies of the ColE1 plasmid inside bacterial cells. Such replication-defective mutant plasmids are activated as a result of suppressor mutations that stabilize stem IV. Thus, the initiation of replication of the ColE1 plasmid depends on the ability of RNA II to form an RNA–DNA hybrid near the origin of replication (ori). At the same time, the formation of a hybrid is influenced by secondary and tertiary structure upstream of the nucleotide sequence of the primer precursor.

Rice. I.48. Scheme of regulation of ColE1 plasmid replication

A– putative secondary structure of RNA II, after transcribing  500 nucleotides of plasmid DNA by RNA polymerase; further elongation of RNA II is accompanied by the formation of a DNA–RNA hybrid (thick arrow) between RNA II and transcribed DNA;

b– a possible mechanism for controlling plasmid replication. The upper part of the figure shows a genetic map of the DNA region necessary for the initiation and control of plasmid DNA replication. The spatial structures of two plasmid replication inhibitors: RNA I and the Rop protein are shown schematically. The lower part shows two alternative conformations of RNA II, formed under the influence of RNA I, I–X - elements of the secondary structure

Control of the number of copies of the ColE1 plasmid. Control of the initiation of replication of the ColE1 plasmid is carried out mainly at the level of changes in the spatial structure of RNA II. Since plasmids control their own biosynthesis, i.e. their replication occurs via an autocatalytic mechanism, it was postulated that the initiation of ColE1 replication is influenced by a plasmid-encoded inhibitor, the concentration of which in the cell is higher, the higher the larger number intracellular copies of the plasmid. Indeed, analysis of the replication mechanisms of mutant plasmids, which are characterized by high copy numbers, made it possible to identify two trance- active factors encoded by the plasmid and influencing the replication of the plasmid in vivo.

The main inhibitor of replication turned out to be a small RNA of 108 nucleotides in length, called RNA I, completely complementary to the 5'-terminal sequence of the primer precursor (RNA II). The promoter of the RNA I gene is located in the region of the origin of replication of the ColE1 plasmid and is directed in the opposite direction to the RNA II promoter (see Fig. I.48). Complementary interactions between RNA I and RNA II influence the formation of the spatial structure of RNA II in such a way that the βγ conformation, inactive with respect to the initiation of replication, preferentially arises (see Fig. I.48, b, bottom right).

The interaction between RNA I and RNA II occurs productively only as long as a short RNA II transcript no longer than 80 nucleotides is synthesized. Although the interaction of RNA I with such a short sequence of nucleotides occurs more slowly than with a transcript 360 nucleotides long, in the latter case RNA I does not affect the conformation of the 5'-terminal part of RNA II and its ability to function as a primer during plasmid replication (conformation αβ, Fig. I.48, b, bottom left). From this it is clear that the rate of formation of hybrids between RNA I and RNA II is decisive for the effective functioning of the mechanism for regulating plasmid replication. The process of interaction between RNA I and RNA II has now been studied in detail. It passes through the formation of several intermediate products and ends with the production of a stable hybrid between RNA I and the 5'-terminal region of RNA II, which are completely complementary to each other.

RNA organizing protein Rop. The gene for the second component, which negatively regulates the replication of the ColE1 plasmid, is mapped directly downstream of the replication origin region. This gene encodes a 63-mer protein called Rop (repressor of primer), which exists in solution as a dimer. Both in vivo and in vitro, Rop enhances the inhibitory activity of RNA I without affecting the synthesis of RNA II. In this case, Rop influences the initial phases of the interaction between RNA I and RNA II, facilitating the transition of the very unstable intermediate product C* to the more stable one – Cm*. The Rop protein has a high affinity for C* and only weakly interacts with isolated RNA I and RNA II in vitro. It is assumed that Rop exhibits minor nucleotide sequence specificity and recognizes some general features the structure of the RNA I–RNA II complex, which arises at the early stages of their interaction. Thus, the functions of the Rop protein apparently consist in converting an unstable RNA–RNA complex into a more stable one, which, in turn, is accompanied by suppression of the formation of the primer necessary for the initiation of replication of the ColE1 plasmid.

The use of antisense RNAs to control the replication of bacterial plasmids is a common technique. In particular, replication of the small, low-copy R1 plasmid is controlled by the RepA protein, which is involved in the initiation of plasmid replication as a positive regulatory factor. RepA synthesis, in turn, is regulated posttranscriptionally by the small antisense RNA CopA, which binds to RepA mRNA in a multistep reaction reminiscent of the hybrid formation between RNA I and RNA II discussed above. This interaction suppresses gene expression repA, possibly due to cleavage of the RNA–RNA duplex by RNase III. The intracellular concentration of antisense CopA RNA is directly proportional to the copy number of plasmid R1. A similar mechanism has been described for the regulation of the initiation of replication of the Staphylococcus aureus plasmid pT181.

When producing bacterial vectors for genetic engineering, many of which contain the origin of replication of the ColE1 plasmid, protein biosynthesis inhibitors, in particular chloramphenicol, are often used to increase the number of their copies in bacterial cells. After discussing the regulatory mechanisms for controlling the replication of this plasmid, the principles on which this technique is based become clear. Indeed, the addition of chloramphenicol to the culture medium blocks the biosynthesis of bacterial proteins, including the Rop protein, which is necessary for effective suppression of the initiation of plasmid replication under the influence of RNA I. As a result, control of the copy number of plasmids in bacterial cells is disrupted, and they begin to replicate continuously using pre-synthesized bacterial proteins for this purpose.

It is known that two phenotypically different plasmids that use the same replication control mechanism are incompatible in the same bacterial cell. Cells containing two plasmids from different compatibility groups quickly form two populations during reproduction, each of which contains only one type of plasmid. This occurs due to the random selection of plasmids for replication within bacterial cells and random distribution the initial pool of plasmids in daughter cells. The evolutionary emergence of a mechanism for controlling the replication of bacterial plasmids using antisense RNAs has expanded the possibilities of the emergence of plasmids belonging to different compatibility groups and coexisting in the same bacterial cells. Indeed, despite the use of the same mechanism, antisense RNAs with different nucleotide sequences will not be able to recognize “foreign,” heterologous RNA targets. This allows such plasmids to coexist in the same bacterial cell and creates conditions for their wider distribution in natural populations of microorganisms.

Lecture 3. Replication of various DNAs and its regulation and repair

Watson and Crick proposed that for DNA to double, the hydrogen bonds holding the helical duplex together must be broken and the strands must separate. They also suggested that each strand of the duplex serves as a template for the synthesis of the complementary strand and, as a result, two pairs of chains are formed, in each of which only one is the parent. Watson and Crick assumed that DNA replication occurs spontaneously, without the participation of enzymes, but this turned out to be incorrect. However, the idea that DNA duplication occurs by combining nucleotides in sequence according to the complementarity rule specified by each strand of the helix solved the conceptual problem of precise gene reproduction.

Since this proposal was made, the template nature of the replication mechanism has been confirmed by numerous data obtained both in vitro and in vivo for various organisms. According to the model, the replication of all double-stranded DNA is semi-conservative. Proof of the semi-conservative mechanism was obtained in 1958 by scientists Meselson and Steel(em). First they grew bacteria long time on a medium containing a heavy nitrogen isotope (15 N), which was incorporated into DNA, and then transferred them to a medium containing a regular light nitrogen isotope (14 N). After replication, the first generation daughter DNA was density fractionated. It turned out that all daughter DNA is homogeneous and has a density intermediate between the density of heavy and light DNA. Consequently, one strand of the daughter DNA molecule contained 15 N, and the other 14 N, which corresponds to a semi-conservative mechanism. Do they exist in nature? alternative ways double-stranded DNA replication (conservative and dispersed) – unknown. So, after one round of replication, one strand in each of the two daughter DNAs is the parent, i.e. conservative, and the other – newly synthesized.

Single-stranded DNA replication in viruses. If the genome is represented by single-stranded DNA (as in some viruses), then this single strand serves as a template for the formation of a complementary strand with which it forms a duplex, and then either daughter duplexes or single-stranded copies of one of their template strands are synthesized on this duplex. Replication of the genetic material of the virus usually occurs with the participation of host cell enzymes. On some viral DNA molecules, DNA copies are also synthesized, using either cellular or virus-encoded DNA polymerase. These DNA copies are subsequently used in the assembly of virus particles. Viral DNA replication occurs either in the nucleus of the host cell (herpes virus) or in the cytoplasm (poxviruses).

Replication in prokaryotes. DNA reduplication(the process by which information encoded in the base sequence of the parent DNA molecule is transmitted with maximum accuracy to the daughter DNA) is carried out by a special enzyme, DNA polymerase. The landing of this enzyme on one of the DNA strands is preceded by a strictly localized break of the ring, if the DNA is circular (in bacteria) and some unwinding of the terminal section of its giant double-stranded helix. Let us immediately note that DNA polymerase can land on either of the two ends of the helix, but always on the strand for which this end is the 3" end (whether it is the “coding” or “protective” strand). Promotion of the enzyme along the “matrix” the mother strand always goes in the direction from the 3" end to the 5" end. It follows that the new DNA strand synthesized using this matrix, “complementary” to it, will begin with its 5" end and grow in the direction of its future 3" - end. These two directions should not be confused. In case of doubt, it is enough to remember that the growth of a newly synthesized thread occurs by sequential addition nucleotides, already carrying a phosphate group bonded to the 5" carbon of deoxyribose. Consequently, it must attach to the previous nucleotide already in place at its OH group bonded to the 3" carbon of deoxyribose. And this means that the growth of a new DNA strand goes in direction 5 "-3" . It is appropriate to recall here that the work of DNA polymerase advancement is carried out due to the energy of breaking the chemical bond between the first and second phosphates of the corresponding nucleoside triphosphate - the precursor of the attached nucleotide.

Now let's move on to additions and clarifications. Let's start with the fact that not one, but three DNA polymerases were found in the E. coli cell. They differ markedly from each other in molecular weight and in the number of molecules of each contained in the cell. And also by their role in the process of DNA reduplication.

Historically the first to be discovered and purified DNA polymerase I(Kornberg enzyme). Then they appeared DNA polymerases II and III, The molecular weights of these three enzymes are, respectively, 109, 90 and 300 kDa, and their representation in one cell is 300, 40 and 20 pieces. The difference in functions will be clear from what follows.

We begin the description of the 1st stage of reduplication with the fact that the initial unwinding of the end of the double-stranded parent DNA molecule is carried out using a special protein "topoisomerases".(transparent 6) at the point of origin of replication - ori (origin - beginning of replication).

Structure of replication origins. DNA fragments carrying the origin of replication have been isolated from E. coli and some plasmids, as well as from yeast and a number of eukaryotic viruses. In some cases, the origin of replication has such a nucleotide sequence that the duplex takes on an unusual configuration, which is recognized by proteins involved in initiation. The nature of the interaction between the origin of replication and proteins and the initiation mechanism in general have been little studied.

So, topoisomerase moves along a double-stranded molecule, weakening its hydrogen bonds so much that in the short terminal section it passes through, these bonds are broken already at a temperature of 37°C. Following topoisomerase, another protein lands on the maternal DNA and begins to move along it. DNA helicase, who will play his role later. Then a special RNA polymerase that works only from the end of the DNA strand, called "primaza" builds a very short chain of ribonucleotides (called "primer") complementary to the beginning of the DNA strand. In bacteria this is only 5 nucleotides, and in eukaryotes it is about 40. (In Fig. 28, all primers are shown as a thin line, and all DNA strands are shown as a bold line.)

Only now, immediately after the primer, DNA polymerase sits on the same DNA strand (for simplicity, let’s call it “first”), which can begin building a complementary DNA strand only starting from the primer, joining it (“dancing from the stove”) . This is DNA polymerase III, the largest, consisting of 6 subunits and the main one in its function - it will conduct “complementary synthesis” of DNA along this first maternal DNA strand until the very end. The initial movement of this DNA polymerase is limited to 1-2 thousand nucleotides of the first strand (in eukaryotes - only 200 nucleotides).

The second mother thread (still empty) forms a “reduplication fork” together with the first thread.

Between the helicase and DNA polymerase III, a certain area of exposed 1st strand is formed. The 2nd thread is also not covered by anything yet. These two threads can close again after the helicase leaves. To prevent this from happening, four so-called "DNA-binding protein". They are not credited with other functions than protection against the restoration of the DNA double helix near the apex of the fork...

Having reached the top of the fork of the diverged strands of maternal DNA, the tandem helicase-DNA-binding proteins - DNA polymerase III stops (see Fig. 28). Topoisomerase moves further along the double-stranded parent DNA, and helicase breaks the sugar-phosphate bond on the 2nd strand. Condensed in the area adjacent to the fork, the turns of the double helix straighten out, the 1st strand of DNA, together with the proteins sitting on it, rotates around its axis, and the cut piece of the 2nd strand, temporarily associated with the helicase, also rotates around this strand. This piece is called the “Okazaki fragment” - after the scientist who discovered the appearance of such fragments during reduplication. Once the tension is removed, the strands of the maternal DNA double helix may begin to separate again. But before that, from the cut end of the Okazaki fragment, another primase begins to build a new ribonucleotide primer on it. Then helicase releases the fragment and moves forward, and a special enzyme "ligase" sews the beginning of the Okazaki fragment to its original place - to the 2nd strand of maternal DNA. Note that ligase (M=96 thousand) in the E.coli cell is represented by the most numerous population - about 200 molecules. From which it follows that it does not perform random “repair” work, but is a full member of the set of enzymes that ensure DNA reduplication (similar to the importance of threads for a surgeon).

When the primer is ready, DNA polymerase I sits in front of it, towards the 5" end of the 2nd mother strand of DNA. The construction of a strand complementary to this fragment of the 2nd strand begins, again in the direction 3" - 5", counting DNA polymerase I reaches the end of the Okazaki fragment and is removed. This ends the 1st stage of reduplication (Fig. 28).

Meanwhile, the primer remaining at the beginning of the 1st strand is destroyed by a certain “ribonuclease H,” an enzyme that breaks the RNA strand complexed with the DNA strand. In its place, DNA polymerase II puts the “correct” deoxyribonucleotides. At the same time, topoisomerase, helicase, and after them DNA polymerase III move forward.

The 2nd stage of reduplication begins. The replication fork also moves forward, the adjacent section of maternal double-stranded DNA becomes compacted and the entire synthesizing tandem stops. Helicase cuts the 2nd strand again, forming a second Okazaki fragment. Just as before, a primer is created at the (temporarily) cut end of the fragment, DNA polymerase I is “attached” to it and begins to copy the second Okazaki fragment, i.e. 2nd strand of maternal DNA. The only difference in the second stage will be that on the path of this polymerase it will encounter a primer left over from copying the 1st Okazaki fragment. But DNA polymerase I, unlike all other DNA polymerases, also has 5"-3" exonuclease activity, i.e. in the direction of its movement. She destroys the primer and reaches the place where her predecessor began copying the 1st fragment of Okazaki. All that remains is to connect these two pieces of the newly synthesized complementary strand with a phosphodiester bond. Naturally, this is done by the ubiquitous DNA ligase.

DNA replication: a tutorial. Regulation of DNA replication Molecular mechanism of replication

History of the study

General views

History of the study

General views

Molecular mechanism of replication

Notes

Literature

See what “DNA replication” is in other dictionaries:

4.2.1.Initiation of DNA replication in E. coli and its regulation

4.2.2.Regulation of ColE1 plasmid replication

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