DNA replication. Replication enzymes

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 area oriC. 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 beginning 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 on oriC. 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. By using electron microscope the parent complex is found as a compact ellipsoidal structure containing 20 DnaA monomers, which closes 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 replication origin regions 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.

Role of oriC topology in 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.

^ Role of proteinDnaAin 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, when elevated temperatures on rich nutrient media) must initiate replication of chromosomes destined for subsequent divisions before the 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 dimer of the bacterial chromosome 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 plasmid replication initiation are based on unique property RNA II forms 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 in the conformation 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). In this case, the formation of the hybrid is influenced by the secondary and tertiary structures 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 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 greater the number of 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 structural features of the RNA I–RNA II complex that 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 the random distribution of the initial pool of plasmids among 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.

The main functional significance of the DNA replication process is to supply the offspring with genetic information. To ensure the genetic stability of an organism and species, DNA must be replicated completely and with very high accuracy. The process of DNA replication is very complex. Many enzymes are involved in it. The first enzymological study of DNA replication was carried out by Arthur Kornberg, who discovered an enzyme in Escherichia coli, now called DNA polymerase I. This enzyme exhibits several types of enzymatic activities and is characterized by a complex structure. DNA polymerase I uses deoxyribonucleoside triphosphates derived from adenine, guanine, cytosine and thymine as substrates. The polymerase activity first demonstrated for DNA polymerase I is characteristic of other polymerases in prokaryotic and eukaryotic cells, but it is important to remember that the main function of E. coli DNA polymerase 1 has been found to be DNA repair.

Initiation of DNA synthesis

To initiate DNA synthesis (Fig. 38.13), short (10-200 nucleotides) RNA sequences that act as primers are required. Synthesis begins with a reaction between the 3-hydroxyl group of the RNA primer and the α-phosphate group of the deoxyribonucleoside triphosphate, during which the deoxyribonucleoside residue is added to the RNA primer with the simultaneous release of pyrophosphate. The 3-hydroxyl group of the attached deoxyribonucleoside monophosphate further carries out a nucleophilic attack on the a-phosphate group of the next inserted deoxyribonucleoside triphosphate, also with the elimination of pyrophosphate. Naturally, the choice of the next nucleotide at each step of synthesis is determined by the DNA template strand according to the rules first proposed by Watson and Crick (Fig. 38.14). Thus, if an adenine deoxyribonucleoside monophosphate residue is located in the corresponding position of the template chain, then thymidine triphosphate will react and its a-phosphate group will be attacked by the 3-hydroxyl group of the last residue of the growing chain. The reaction occurs only if the inserted nucleotide forms a complementary pair with the next nucleotide of the DNA template chain and, due to hydrogen bonds, occupies a position in which the 3-hydroxyl group of the growing chain attacks the new nucleotide and incorporates it into the polymer. The DNA sequences attached to RNA primers were named after the Japanese scientist who discovered them - Okazaki fragments (Fig. 38.15). In mammals, after the formation of a significant number of Okazaki fragments, the replication complex begins

(see scan)

Rice. 38.13. Initiation of DNA synthesis by an RNA primer and subsequent addition of a second deoxyribonucleoside triphosphate.

Rice. 38.14. Template function of the DNA strand during RNA primer-initiated synthesis of the complementary strand.

Rice. 38.15. Discontinuous polymerization of deoxyribonucleotides and formation of Okazaki fragments.

to the removal of RNA primers and filling the resulting gaps with the corresponding deoxyribonucleotides. Then, using DNA ligase, the fragments are “stitched” together to form a continuous DNA chain.

Replication polarity

As already noted, DNA molecules consist of two antiparallel chains. DNA replication in pro- and eukaryotes occurs simultaneously on both strands. However, there is no enzyme that leads DNA synthesis in the direction, and, therefore, the newly synthesized chains, it would seem, cannot grow in the same direction at the same time. Despite this contradiction, the same enzyme carries out almost synchronous synthesis of both chains. In this case, the chain synthesized in the 5-U direction (“leading”) turns out to be continuous. The synthesis of the second (“lagging”) strand is carried out in fragments of 150-200 nucleotides. The next acts of initiation of the synthesis of these fragments, which occurs at any given moment, are also carried out in the direction as the replication fork generally moves in one direction. The scheme of “semi-continuous” DNA synthesis is shown in Fig. 38.16.

During the replication of mammalian nuclear DNA

Rice. 38.16. The process of semi-continuous, simultaneous replication of both strands of double-stranded DNA.

Most of the RNA primers are removed at the end of the process, meanwhile, during replication of the mitochondrial genome, small fragments of RNA remain integrated in a closed circular DNA molecule.

DNA polymerization and repair enzymes

In the nucleus of mammalian cells there is a class of polymerases, the so-called polymerases alpha (Pol a), responsible for chromosomal replication. One Pol a molecule can incorporate about 100 nucleotides per second into a growing chain, so it functions about ten times slower than bacterial DNA polymerase. The decrease in speed may be due to interference from nucleosomes. How DNA polymerase overcomes nucleosomes is unknown. However, it is known that after replication is completed, the corresponding nucleosomes are distributed randomly in both daughter strands.

In the nuclei of mammalian cells, a DNA polymerase with a smaller molecular weight than Pol a is also found - polymerase beta (Pol, which is not involved in the normal replication process, but is necessary for DNA repair (see below). Another, mitochondrial, DNA -polymerase gamma (Poly) carries out replication of the circular genome of mitochondria.

Complete replication of the mammalian genome is completed in 9 hours—the time required to double the genetic material of a diploid dividing cell. This speed indicates that replication begins at many sites at once, called replication origins and designated ori (from the English origin - beginning). There are about 100 such points (sites). Replication occurs in two directions, and both chains are replicated simultaneously. In this case, so-called “replication bubbles” are formed on the chromosome (Fig. 38.17).

The sites that act as origins of replication in eukaryotes are not clearly defined. More complete data in this regard are available for yeast and several animal viruses. We can say with confidence that the initiation processes are controlled both spatially and temporally, since neighboring clusters are initiated synchronously. It is believed that functional chromatin domains are replicated as integral units. This implies that the origins of replication are located in a very specific manner relative to the transcription units.

Rice. 38.17. Formation of “replication bubbles” during DNA synthesis. The bidirectionality of replication and the proposed location of strand unwinding proteins in replication forks are shown.

Rice. 38.18. Hypothetical scheme of action of a protein specific to single-stranded DNA at the replication fork. As the second strand is synthesized, the protein is released and attached to the newly formed sections of single-stranded DNA. (Courtesy of V. Alberts.)

(see scan)

Rice. 38.19. Comparison of two types of reactions that repair single-strand DNA breaks. The reactions shown on the left are catalyzed by DNA ligase, while those shown on the right are catalyzed by DNA topoisomerase. (Slightly modified and reproduced, with permission from Lehninger A. L.: Biochemistry, 2nd ed. Worth, 1975.)

During replication, double-stranded DNA must separate into individual strands so that each of them can function as a template. The separation of DNA chains is facilitated by molecules of specific proteins that stabilize the single-stranded structure as the replication fork advances. Stabilizing proteins bind stoichiometrically to a single strand without interfering with the nucleotides acting as a template (Fig. 38.18). Along with the separation of the chains, the unwinding of the helix should also occur (1 turn for every 10 nucleotides), accompanied by the twisting of the newly synthesized daughter chains. Considering the time it takes for replication to occur in prokaryotes, it can be calculated that the DNA molecule should unwind at a speed of 400,000 rpm, which is completely impossible. Therefore, there must be multiple "hinges" located along the entire length of the DNA molecule. Hinge functions are performed by a special enzyme (DNA topoisomerase), which introduces breaks in one of the chains of the unwinding double helix. The breaks are quickly repaired by the same enzyme without additional energy costs, since the necessary energy is stored in the form of a high-energy covalent bond that occurs between the sugar-phosphate backbone of the DNA chain and topoisomerase. Shown in Fig. 38.19 The diagram of this process can be compared to the sequence of DNA ligase-catalyzed DNA ligase stitching events. DNA topoisomerases are also responsible for unwinding supercoiled DNA. Supercoiled DNA is a highly ordered structure formed by circular or ultra-long DNA molecules when twisted around a histone core (Fig. 38.20).

One class of animal viruses (retroviruses) has enzymes capable of synthesizing a DNA molecule from an RNA template. RNA-dependent DNA polymerase, or reverse transcriptase (this is the name of this enzyme), first synthesizes an RNA-DNA hybrid using the ribonucleic acid genome of the virus as a template. The RNase H enzyme then removes the RNA strand, and the remaining DNA strand in turn serves as a template for the synthesis of the second DNA strand. Thus, a cDNA double-stranded DNA copy appears, containing information primarily presented in the form of the RNA genome of the retrovirus.

Regulation of DNA synthesis

In animal and human cells, DNA replication occurs only during a certain period of the cell's life. This period is called synthetic (the so-called -phase). -phase is separated from mitosis by presynthetic and postsynthetic periods (Fig. 38.21).

Rice. 38.20. DNA supercoiling. A left-handed toroidal (solenoid) superhelix (left) turns into a right-handed one when the cylindrical core is removed. A similar transition occurs when nucleosomes are destroyed in the case of histone extraction with concentrated saline solutions.

The primary regulation of the cell's own DNA synthesis is that replication occurs at a strictly defined time and mainly in cells preparing for division. The regulation of cell entry into the -phase involves cyclic purine nucleotides and, possibly, the substrates of DNA synthesis themselves. The mechanism of such regulation remains unknown. Many oncoviruses are capable of weakening or destroying internal information connections that control the entry of cells into the -phase. Again, the mechanism remains unclear, although it may involve phosphorylation of certain protein molecules of the host cell.

In the -phase, mammalian cells contain more polymerase a than in non-synthetic periods of the cell cycle. In addition, in the -phase, the activity of enzymes involved in the formation of substrates for DNA synthesis (deoxyribonucleoside triphosphates) increases. The activity of these enzymes decreases upon exiting the -phase and remains at a low level until a signal is received to resume DNA synthesis. In the -phase, complete and strictly single replication of nuclear DNA occurs. It seems that the replicated chromatin is somehow marked, as a result

Rice. 38.21. Mammalian cell division cycle. The DNA synthesis phase (-phase) is separated from mitosis by periods G, and G,. (The arrow indicates the direction of the cell development cycle.)

which interferes with further replication until the cell undergoes mitosis. It can be assumed that methyl groups act as such a covalent marker (i.e. DNA marking is carried out due to its methylation).

As a rule, each given pair of chromosomes replicates simultaneously and during a strictly defined period of the S-phase. The nature of the signals regulating DNA synthesis at this level is unknown, but, apparently, each individual chromosome has such a regulatory mechanism.

DNA degradation and repair

The transmission of hereditary information in an undistorted form is the most important condition for the survival of both each individual organism and the species as a whole. Therefore, during evolution, a system must have been formed that allows the cell to correct DNA damage caused by replication errors or damaging environmental influences. It is estimated that, as a result of damage due to these causes, an average of six nucleotide substitutions occur per year in the genome of human germline cells. Apparently, approximately the same number of mutations occur in somatic cells per year.

As described in Chap. 37, the main condition for accurate replication is the correct formation of nucleotide pairs. The accuracy of complementary interactions depends on whether the purine and pyrimidine nucleotides are in a tautomeric form favorable for pairing (Fig. 34.7). At equilibrium, the concentration of favorable tautomeric forms of nucleotides exceeds the concentration of unfavorable tautomers by 104-105 times. This is clearly not enough to ensure unmistakable recognition. This is why bacterial and mammalian cells have a special system for monitoring the accuracy of nucleotide pairing. This step is checked twice: once when deoxyribonucleoside triphosphates are incorporated into the growing strand, and again after incorporation, using an energy-dependent mechanism to remove erroneously inserted nucleotides from the newly synthesized DNA strand. Thanks to this control, inclusion errors occur no more than once per 108-10 base pairs. In E. coli cells, this mechanism is provided by DNA polymerase activity. At the same time, mammalian DNA polymerases do not have obvious proofreading nuclease activity.

Physical and chemical environmental factors cause four types of DNA damage (see Table 38.2). Damaged areas can be repaired, replaced by recombination, or remain unchanged. In the latter case, mutations occur, potentially leading to cell death. The possibility of repair and replacement is based on the redundancy of information encoded in the structure of double-stranded DNA: a defective region of one DNA strand can be corrected using an intact complementary strand.

The key moment of all recombination and repair events is recognition of the defect, accompanied by either direct repair or marking for subsequent correction. The thermolability of the N-glycosidic bond of purines leads to DNA depurination with a frequency of about 5000-10000 per cell (per day) at 37 ° C. Depurination sites are recognized by special enzymes,

Table 38.2. Types of DNA Damage

specifically filling the gap without breaking the phosphodiester backbone of the molecule.

Both cytosine and adenine bases are spontaneously deaminated to form uracil and hypoxanthine, respectively. Since DNA normally contains neither uracil nor hypoxanthine, it is not surprising that specific α-glycosylases recognize these abnormal bases and remove them. The resulting break serves as a signal for the action of repair purine- or pyrimidine-specific endonucleases, which cleave the phosphodiester bond near the corresponding site of damage. After this, with the sequential action of exonuclease, repair DNA polymerase and DNA ligase, the gap is filled and the original correct structure is restored (Fig. 38.22). This chain of events is called excision repair. The process of repair of DNA containing alkylated bases and base analogues occurs in a similar way.

Repair of deletions and removal of insertions occurs through recombination processes, which can occur either with or without the participation of replication.

Ultraviolet radiation induces the formation of pyrimidine-pyrimidine dimers.

Rice. 38.22. The enzyme uracil DNA glycosylase removes uracil resulting from spontaneous deamination of cytosine. (Courtesy of V. Alberts.)

Rice. 38.23. Thymine-thymine dimer, formed by the binding of adjacent thymine bases.

This process affects predominantly the thymine bases of the same chain located one below the other (Fig. 38.23). There are two mechanisms for removing thymine dimers in the cell: excision repair and photoreactivation. This repair method involves photoactivation by visible light of a specific enzyme, which reverses the process that led to the formation of the dimeric structure.

Single-strand DNA breaks caused by ionizing radiation can be repaired by direct ligation or recombination. The mechanisms involved in the repair of cross-links between the bases of opposing strands or between DNA and proteins are not well understood.

So, repair of damage caused by ionizing radiation and base alkylation is carried out by excision and resynthesis of short sections of DNA. Removal of damage caused by ultraviolet irradiation, as well as cross-links, is achieved in a similar way, but in this case longer sections of DNA are affected. In mammalian cells, the occurrence of repair replication is evidenced by unscheduled DNA synthesis, i.e. inclusion of radioactive precursors outside the S phase into DNA.

With the intensification of excision repair processes in response to damaging effects in mammalian cells, the activity of the enzyme poly(AOP-ribosyl) polymerase increases. This enzyme, with the participation of a coenzyme, carries out the reaction of ADP-ribosylation of chromatin proteins. Mostly mono-ADP-ribosylation occurs, but sometimes homopolymer chains (ADP-ribose) are added. The function of poly(AOP-ribosyl) polymerase or its product (ADP-ribose) in the process of excision repair is not entirely clear. There is a correlation in time between the intensification

repair processes and increased enzyme activity. Specific inhibition of the activity of this enzyme prevents the elimination of breaks in the DNA chain. The increase in poly (ADP-ribosyl) polymerase activity is apparently caused by DNA fragmentation in the nucleus. Such fragmentation can be induced primarily by physical agents (e.g. x-ray radiation), in addition, it may occur inappropriately intensively during the repair of ultraviolet damage or damage caused by alkylating agents. The increase in enzyme activity caused by DNA breaks can be so great that it can lead to depletion of the intracellular supply of the coenzyme NAD+.

Xeroderma pigmentosum is an autosomal recessive hereditary disease. The clinical syndrome involves sensitivity to sunlight (ultraviolet light), which leads to the formation of multiple skin cancers and death. This disease appears to be associated with impaired repair processes. In cultured cells of patients with xeroderma pigmentosum, the intensity of photoreactivation of thymine dimers is reduced. However, the actual genetic disorders leading to xeroderma pigmentosum include at least 7 complementation groups, which indicates the complexity of the causes of this disease.

In juseroderma pigmentosum, cells of most (if not all) complementation groups exhibit abnormalities in the speed and intensity of the poly(AOP-ribosyl) polymerase response to ultraviolet irradiation. In cells of at least one complementation group, the decrease in enzyme activity appears to be associated with an inability to cut the DNA strand at the damaged site, since the addition of deoxyribonuclease to such defective cells normalizes the level of poly(AOP-ribosyl) polymerase activity.

Patients with ataxia telangiectasia (an autosomal recessive disease leading to cerebellar ataxia and lymphoreticular neoplasm) have increased sensitivity to x-ray radiation. Patients with Fanconi anemia (an autosomal recessive anemia associated with an increased incidence of cancer and chromosomal instability) are likely to have an impaired cross-link repair system. All three described syndromes are characterized by an increased incidence of tumors. It is possible that in the near future other human diseases will be identified that are caused by defects in the DNA damage repair system.

LITERATURE

Bauer W. R. et al. Supercoiled DNA, Sci. Am. (July), 1980, 243, 118.

Cantor C.R. DNA choreography, Cell, 1981, 25, 293. Igo-Kemenes T., Horz W., Zachau H.G. Chromatin, Annu.

Rev. Biochem., 1982, 51, 89.

Jelinek W. R.. Schmid C. W. Repetitive sequences in eukaryotic DNA and their expression, Annu. Rev. Biochem., 1982,51,813.

Jongstra J. et al. Induction of altered chromatin structures by simian virus 40 enhancer and promoter elements, Nature, 1984, 307, 708.

Kornberg A. DNA Replication, Freeman, 1980.

Lindahl T. DNA repair enzymes, Annu. Rev. Biochem., 1982, 51, 61.

Loeb L. A., Kunkel T. A. Fidelity of DNA synthesis, Annu. Rev.

Biochem, 1982, 51, 429.

McGhee J. D., Felsenfeld G. Nucleosome structure, Annu. Rev.

Biochem., 1980, 49, 1115.

Nossal N. G. Prokaryotic DNA replication systems, Annu. Rev. Biochem., 1983, 52, 581.

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 for a long time on a medium containing a heavy isotope of nitrogen (15 N), which was incorporated into DNA, and then transferred them to a medium containing a regular light isotope of nitrogen (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. Whether alternative methods of double-stranded DNA replication (conservative and dispersed) exist in nature is 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 be added 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 again cuts the 2nd strand, 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.

ribonuclease H

DNA polymeraseII

Meanwhile, in the region where the third apex of the reduplication fork is formed, exactly the same events occur as at the 2nd stage of reduplication. The rate of this process is estimated to be approximately 1000 nucleotides per second in bacteria, 100 in animals and 20 in plants.

It is very likely that at the same time, similar processes of unwinding the double helix with the formation of Okazaki fragments and the complementary construction of new DNA strands also occur from the opposite end of the maternal DNA. Of course, there DNA polymerase III continuously moves along that DNA strand, which we called the 2nd, and the 1st strand is cut into Okazaki fragments. When the two movements meet, two daughter copies of the original DNA are ready. (They will be “stitched” together by the same ligation.) By the way, it turned out that the length of Okazaki fragments in E. coli (1-2 thousand nucleotides) is significantly longer than in eukaryotes (less than 200). It is not without interest that this last figure coincides with the length of DNA in the nucleosome (see below).

A more complex model of replication fork movement involves the formation of a replisome, a multienzyme complex of a higher level of organization. This complex consists of a functional primemosome-primase complex, helicase, polymerase III, and possibly gyrase. Such a complex can provide elongation of the leading strand and, at the same time, initiation of primer RNA, as well as completion of DNA during the synthesis of the lagging strand. Two replisomes working in concert at two replication forks that move in opposite directions along a circular chromosome would make this model even more elegant.

Replication of circular duplexes. Replication is also initiated at the origin of replication (ori).

Growing strands form replication forks that cross in either two (top) or one (bottom) directions, depending on the nature of the origin of replication.

In some circular genomes, each strand has its own origin of replication (for example, in animal mitochondrial DNA). The synthesis of one chain begins at the point ori >R >. When the new chain reaches the point ori >D >, the synthesis of another chain begins. Synthesis is initiated by the formation of primer RNA.

Some double-stranded ring chromosomes replicate in an alternative manner called rolling circle replication. In this case, double-stranded circular DNA is cut by a specific enzyme at a unique site on one chain (the starting point of the rolling ring), and nucleotides are added to the resulting 3΄ hydroxyl ring using polymerase III; in this case, the matrix is an intact closed circuit. Thus, only the leading strand is synthesized in the fork. As the chain synthesizes, the 5΄ end of the nicked ring is displaced as a single chain. As a result, the length of the leading chain can exceed the length of the matrix by 2-5 times. This method of replication is used by phages M13 or fX174 (their mature genomes are single-stranded circular DNA) in the late stages of the infectious process, after the infecting DNA is converted into a double-stranded circular form. The continuously separating single strands of DNA produced by rolling circle replication are cut at each origin of replication and closed to form mature forms that are packaged into viral particles. Phage λ uses this mode of replication to form double-stranded linear viral DNA. The substrate matrix in this case is double-stranded circular DNA, which was replicated after linear viral DNA was converted into a circular replicative form early in the infection.

As for the reduplication mechanism in eukaryotes, although it is less studied, as many as five DNA polymerases have been found and characterized here, which are usually designated by Greek letters: α, β, γ, δ, and ε. The main enzyme similar to DNA polymerase III in bacteria is DNA polymerase δ. DNA polymerase α is responsible for constructing primers (from ribonucleotides). DNA polymerase β - copies Okazaki fragments and is responsible for DNA repair. DNA polymerase γ carries out DNA synthesis in mitochondria. The function of DNA polymerase ε is still unknown.

Of course, the giant DNA of higher organisms begins reduplication not only from the ends of the molecule, but also at many intermediate points. It is believed that in yeast there are about 300 such origins of replication. They are separated from each other by 40 thousand nucleotide pairs. There are up to 20,000 start points in human DNA, located at intervals of 150 thousand base pairs. Apparently, the places where DNA polymerase δ begins unwinding and landing are sequences of relatively weakly bound A-T steam grounds. After initiation, replication continues in two directions from each point until the replication forks of two adjacent replication origins merge. The full-length DNA of each daughter chromosome is obtained by joining shorter, independently initiated, newly synthesized strands.

Telomeres and centromeres. Centomeres and telomeres are the most clearly defined morphological structures of chromosomes (transparency 12). For a long time it was believed that their structure and functions are associated with some special DNA sequences. However, it was possible to identify only one such feature at the molecular level: the presence in the region of centromeres and telomeres satellite DNA. Satellite DNA is long tandem repeats located in the centromere and telomere regions.

Structure of centromeres. In mammals, centromeres have a complex disc-shaped structure called a kinetochore. There is one kinetochore disk on each side of the chromosome. During mitosis, spindle fibril microtubules attach directly to the dense outer layer of the kinetochore, associated with chromatin loops. Kinetochores in yeast form CEN regions (short DNA segments) together with DNA-binding proteins (transparent 13). Sequences located on one or both sides of the CEN regions can block the passage of the replication fork until a specific signal appears that allows replication to terminate in anaphase. In this case, the number of chromosomes will not exceed one per daughter cell.

Sequences in the telomere region. Telomeres, the ends of a eukaryotic chromosome, are also the ends of a linear DNA duplex. One of the replication problems is associated with telomeres: how are the 5΄ ends of a chromosomal duplex completed if DNA polymerases do not initiate the synthesis of new chains? Perhaps this issue is resolved in the same way as during the replication of a linear duplex of adenoviruses, or using alternative mechanisms? Recent evidence suggests that the terminal regions of eukaryotic chromosomes—telomeres—replicate through a special mechanism. The ends of the chromosomes of yeast, invertebrates, plants and vertebrates have a similar structure: they contain hairpin-like structures in which the 3΄ and 5΄ ends of the DNA duplex are adjacent, and many tandem repeats. There are multiple single-strand breaks near a loop in one of the strands in the repeat region. Recently from Tetrahymena an enzyme was isolated - telomerase - terminal deoxynucleotidyl transferase, which attaches the 5΄-TTGGGG-3΄ repeat, sequentially one nucleotide at a time, to the 3΄ ends of specific oligonucleotide primers (TTGGGG) >n > ( Tetrahymena) and (TGTGTGGG) >n > (yeast). Thus, telomerase can build telomeres without using parental DNA as a template (transparent 20). Telomerase is a large ribonucleoprotein complex, and its enzymatic activity requires both RNA and proteins. A hypothetical diagram of telomere formation is shown in the figure. The upper part of the figure shows the formation of a loop at the end of the chain containing the sequence 5΄-(TTGGGG) >n >-3΄, and single-strand breaks on the opposite strand containing the sequence 5΄-(CCCCAA) >n >-3΄. To the 3΄-end of the lower chain, 5΄-TTGGGG-3΄ units are added sequentially, one nucleotide at a time, using teloisomerase. Primase and DNA polymerase copy the 5΄-(TTGGGG) >n > -3΄ strand to form new 5΄-(CCCCAA) >n > -3΄ units. As a result of incomplete ligation, single-strand breaks remain in the C-rich strand. At the 3΄ end of the 5΄-(TTGGGG) >n >-3΄ chain, a loop is again formed, stabilized by interactions between guanosine residues.

Termination of replication.

Termination and divergence in circular genomes. The closed structure of many genomic DNA simplifies the process of completing replication of the entire nucleotide sequence. Continuous growth of the leading and lagging strand along the circular template inevitably leads to the alignment of the 3΄-hydroxy and 5΄-phosphoryl ends of one chain either at the origin of replication or, in bidirectional replication, in the middle of the ring (transparent 14). The rings at these meeting points are connected by DNA ligase, and they usually end up linked in pairs, and subsequently they must be separated into separate genomes. This occurs via topoisomerase type II (transparent 15).

Termination and divergence in linear DNA. With the exception of adenoviral DNA, where the synthesis of DNA strands is initiated by a protein primer and the template strand is copied in its entirety (transparent 16), in all other cases an RNA primer is required for replication, which creates special problems when completing the replication of linear duplex DNA (transparent 17). The fact is that after the initiation of the synthesis of a new chain and the subsequent removal of the RNA primer, the newly synthesized chain contains a gap at the 5΄ end. Since there is no way to extend the 5΄ ends of DNA strands, some other methods of completing replication are needed. Were suggested two ways.

First: suggests that there are DNA strands with direct repeats at the ends (Clear 18). After replication, the two complementary ends of both incomplete duplexes can pair and form linear concatemers with single-strand breaks. The remaining gaps can be filled by extending the strands in the 3΄ → 5΄ direction and then joining them with DNA ligase or by directly joining the docking ends with DNA ligase. ligases with the formation of concatemers. After cutting the concatemer with a specific endonuclease, overhanging 5΄ ends are formed, and DNA polymerase can extend shorter chains from the 3΄ end.

Second. It assumes the presence of short inverted repeats at the end of each DNA strand, due to which small loops are formed (transparent 19). The 3΄ end of the loop serves as a primer for copying the unreplicated region. Due to a specific break at the beginning of the inverted repeat, a structure is obtained that can be completed from the 3΄ end to restore the original double-stranded terminal sequence.

Let us also note that all DNA polymerases that conduct complementary synthesis, both in bacteria and eukaryotes, also have exonuclease activity in the 3"-5" direction, the opposite direction of synthesis. They are able to “turn around” and remove the nucleotide they just attached. This is a very important mechanism for eliminating errors in complementary synthesis. After all, a mistake can only be discovered when it has already been made. And fix it immediately! DNA polymerases “can” do this. Such a correction is not uncommon, but the norm. It is believed that without it, during DNA reduplication from E. coli, 5-10% of the nucleotides would be attached incorrectly. Thanks to correction, there is 1 error in this DNA per ten million base pairs.

DNA repair

DNA is the only cell macromolecule that is capable of eliminating (repairing) damage that occurs in its structure. Moreover, it encodes information about the mechanisms of a wide variety of repair processes. Complementary pairing underlies not only DNA replication, but also the process of restoring the original DNA structure during repair of damage affecting the backbone of the molecule, modification of a particular base, or mispairing during recombination (see below). Simultaneous damage to both strands in one place and double-strand breaks are often lethal to DNA, since such defects are repaired only in rare cases.

Most often, the glycosidic bonds between purine and deoxyribose N are broken (depurination) with increasing temperature. Every day, from 5,000 to 10,000 acts of depurination occur in a human cell - this leads to disruption of gene replication and expression. In addition, C and A residues can undergo spontaneous deamination to form Y and hypoxanthine residues, respectively; the frequency of such events is approximately 100 per genome - this leads to the appearance of mutations.

Many changes in DNA structure occur under the influence of chemicals present in the environment. These are alkylating agents (nitrogenous compounds, alkyl sulfonates, nitrosoureas), which preferentially modify G-residues, compounds that are inserted between adjacent base pairs and lead to the appearance of insertions and deletions during replication; bifunctional agents capable of forming covalent crosslinks between two DNA strands and blocking their divergence during replication. No less destructive physical influences– absorption of T or C UV light leads to the formation of cyclobutane dimers between neighboring pyrimidines; ionizing radiation (cosmic rays) promotes the formation of highly reactive free radicals, which have a wide variety of effects on DNA; X-rays cause single- and double-strand breaks in DNA, as well as other damage characteristic of free radicals.

There are two main methods of repair processes:

direct correction of modifications or mismatches without requiring replication to restore the original structure;

removal of nucleotides surrounding mismatched or altered base pairs and resynthesis of this region by replication.

repair by direct restoration of the original structure.

If, under the influence of alkylating agents - N-methyl-N-nitrosourea or N 1, N-dimethylnitrosoguanidine, O 6 - methyl- or O 6 -alkyl-substituted guanine residues are formed in DNA, then dealkylation of such residues occurs with the participation of enzymes - O 6 -methylguanine -DNA alkyltransferase, which catalyzes the transfer of alkyl groups to sulfhydryl groups of cysteine residues of the enzyme, while the acceptor protein is inactivated. This occurs in bacteria and mammals.

If, upon irradiation of DNA with UV light, cyclobutane dimers are formed between adjacent pairs of pyrimidine bases, then they are converted enzymatically (photolyases are not present in mammals) into monomers when the solution is illuminated with visible light in the wavelength range of 300-600 nm. The enzyme forms a stable complex with the pyrimidine dimer and, using the energy of the light absorbed by it, destroys the dimer without breaking the DNA strands.

Repair by replacement of modified residues.

Replacing a modified nucleotide usually occurs in four steps:

Stage 1. The enzyme recognizes this nucleotide and cuts the polynucleotide chain near it or breaks the glycosidic bond between the modified base and deoxyribose. Cleavage of sites where depurinization or depyrimidinization has occurred is carried out by AP (apurinic, apyrimidinic) endonucleases. In the repair of N-alkylated purines and other modified bases, a key role is played by enzymes - N-glycosylases, which cleave the glycosidic bond between the modified base and desoskiribose (transparency 25)

Stage 2. The exonuclease removes the modified nucleotide and/or neighboring nucleotides, leaving a small gap.

Stage 3. The deleted region is synthesized anew from the 3΄-OH-terminus using the opposite strand as a template.

Stage 4. The ends of the break formed as a result of repair are connected to restore the covalent integrity of the repaired chain (transparent 26)

The importance of DNA repair.

Elimination of replication errors is important, since most of the damage blocks the transfer of genetic information to the next generation, and the rest, if not eliminated, will remain in the genome of descendants and lead to dramatic changes in the molecules of proteins and enzymes necessary to maintain the life of the cell. When certain parts of the repair system are damaged, cells become especially vulnerable to certain chemical and physical agents. People suffering from xeroderma pigmentosum, for example, are very sensitive to UV light and develop various forms of skin cancer even with very little exposure to sunlight. The cells of such people carry a mutation in the RAD genes, which manifests itself in the fact that their ability to cleave pyrimidine dimers from UV-irradiated DNA is impaired. The disease can be caused by a mutation in one of at least nine genes, which suggests a fairly complex mechanism for repairing DNA containing thymine dimers in humans. Typically, the disease is associated with an inability to clear thymine dimers. If an enzyme with thymine dimer glycosylase and AP endonuclease activities is added to irradiated cells in culture, then UV damage can be eliminated.

DNA replication

Schematic representation of the replication process:(1) lagging strand, (2) leading strand, (3) DNA polymerase, (4) DNA ligase, (5) RNA primer, (6) primase, (7) Okazaki fragment, (8) DNA polymerase , (9) helicase, (10) single strand with associated proteins, (11) topoisomerase.

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 the replisome .

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.

Replicon is a section of DNA that contains the replication initiation site and is replicated after DNA synthesis begins from this site.

Replication begins at the replication initiation site with the unwinding of the DNA double helix, which forms replication fork- site of direct DNA replication.

The essence of DNA replication is that a special enzyme breaks the weak hydrogen bonds that connect the nucleotides of the two chains. As a result, the DNA strands are separated, and free nitrogenous bases “stick out” from each strand (the appearance of a so-called replication fork). A special enzyme DNA polymerase begins to move along the free DNA strand from the 5" to the 3" end (leading strand), helping to attach free nucleotides constantly synthesized in the cell to the 3" end of the newly synthesized DNA strand. On the second strand of DNA ( lagging thread) new DNA is formed in the form of small segments consisting of 1000-2000 nucleotides (Okazaki fragments).

To begin the replication of DNA fragments of this strand, the synthesis of short RNA fragments as seeds is required, for which a special enzyme is used - RNA polymerase (primase). Subsequently, the RNA primers are removed, and DNA is inserted into the resulting gaps using DNA polymerase I. Thus, each DNA strand is used as a template or template for constructing a complementary strand.

Main DNA replication enzymes:

DNA polymerase

DNA polymerase is an enzyme involved in DNA replication. Enzymes of this class catalyze the polymerization of deoxyribonucleotides along a chain of DNA nucleotides, which the enzyme “reads” and uses as a template. DNA polymerase begins DNA replication by binding to a stretch of nucleotide chain.

DNA ligases

Ligase is an enzyme that catalyzes the joining of two molecules to form a new chemical bond (ligation). DNA ligases are enzymes that catalyze the covalent cross-linking of DNA strands during replication.

DNA helicases

DNA helicases are enzymes that unwind the double-stranded DNA helix.

DNA topoisomerases

DNA topoisomerases are enzymes that change the degree of superhelicity and the type of superhelix. By means of a single-strand break, they create a hinge around which the unreplicated duplex of DNA located in front of the fork can rotate freely. This takes off mechanical stress, which occurs when two chains unwind in a replication fork, which is a necessary condition for its continuous movement.

Primaza

Primase is an enzyme with RNA polymerase activity; serves for the formation of RNA primers necessary for the initiation of DNA synthesis at the ori point and further for the synthesis of the lagging strand.