Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Organization of the Cellular Genetic Apparatus
Nucleic Acid Replication

The primary function of DNA is its ability to self-replicate. Replication is a highly precise mechanism that is virtually error-free. DNA itself (or RNA in some Viruses) encodes The Structure of the Enzymes responsible for doubling Nucleic Acids, synthesizing new NUCLEOTIDES—the Building Blocks of replication—correcting replication errors, and repairing DNA Damage caused by various factors. Ultimately, the very structure of DNA, specifically its double-stranded nature, facilitates the copying process because each strand can act as a template for synthesizing new DNA molecules. This hypothesis was proposed by James Watson and Francis Crick as early as 1953 and has since been experimentally confirmed. Such a mechanism of DNA copying, where each strand serves as a template and the newly synthesized molecules are hybrid (consisting of one old and one new strand), is called semi-conservative.

In addition to the semi-conservative model, two other Replication Models were proposed: conservative and dispersive. The characteristics of these METABOLISM/36.html">DNA replication models are as follows. According to the dispersive model, the parental DNA helix breaks apart at each half-turn through multiple fragmentation during doubling, and the synthesis of new strands occurs on these fragments (Fig. 1.9). In the conservative model, the DNA helix does not unwind at all and serves as a template for two entirely new strands, resulting in a parental helix composed entirely of old material and a daughter helix composed of new material. The validity of the semi-conservative DNA Replication Mechanism was proven by Meselson and Stahl in 1958 through ultracentrifugation experiments using labeled bacterial DNA.

The Essence of these experiments was as follows: E. coli DNA was labeled with the radioactive isotope ¹⁵N and then allowed to undergo one round of replication by growing the Cells for ~50 minutes in a nutrient medium containing the normal nitrogen isotope, ¹⁴N. DNA extracted from the cells was subjected to cesium chloride density gradient ultracentrifugation. In this type of centrifugation, CsCl molecules establish a density gradient in the tube, and molecules of other substances distribute themselves within this gradient according to their density. The DNA of E. coli grown on a medium containing ¹⁵N has a density of 1.724 g/cm³, whereas the DNA of cells grown on a conventional medium with the ¹⁴N isotope has a density of 1.710 g/cm³. Thus, a mixture of these Two Types of DNA is easily separated by density centrifugation. The localization of DNA in the CsCl gradient tube can be determined by ultraviolet Light absorption (DNA absorbs radiation at a wavelength of 260 nm). Consequently, DNA is detected in the tube as "bands"—the "light" band near the top of the tube and the "heavy" band closer to the bottom. In this experiment, only a single, intermediate-density band formed in the cesium chloride gradient tube, THE POSITION OF which corresponded to hybrid DNA containing both nitrogen isotopes, ¹⁵N and ¹⁴N. This circumstance ruled out one of the DNA replication models—the conservative model. To distinguish between the two remaining replication models, Meselson and Stahl allowed the Bacteria, whose DNA contained both isotopes, to undergo another Cell Division in a ¹⁴N medium. Their DNA was then subjected to ultracentrifugation once again. This time, two DNA bands formed in the tube—a "light" one and an intermediate-density one—confirming the validity of the semi-conservative DNA replication mechanism.

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Fig. 1.9. Proposed models of duplex DNA replication. Solid lines represent the original ("heavy," ¹⁵N-containing) DNA strands, and dashed lines show the new ("light," ¹⁴N-containing) DNA strands resulting from replication.

Thus, all nucleic acid replication Methods studied to date adhere to the semi-conservative mechanism, whereby after each round of replication, one strand in each of the two daughter molecules is parental (conservative) and the other is newly synthesized. The replication of single- and double-stranded nucleic acids representing the genomes of various organisms follows certain regularities across different mechanisms, as discussed below. Common to all these processes are: 1) the involvement of a complex enzymatic machinery that carries out replication; 2) the presence of three main stages—initiation, elongation, and termination; 3) adherence to THE PRINCIPLE OF complementarity during the construction of new strands, using the parental strand as a template; 4) high process fidelity; and 5) The ability to correct replication errors via proofreading.

Replication of double-stranded DNA. Double-stranded DNA constitutes the genomes of all cellular organisms, both PROKARYOTES AND EUKARYOTES. The Mechanism of DNA replication is best understood in Prokaryotic Cells, particularly the bacterium E. coli. Experiments with prokaryotes have shown that under conditions limiting Protein Synthesis, DNA replication does not occur, leading to the Conclusion that this process requires protein participation. It has now been demonstrated that products of more than 10 genes are involved in DNA replication. These primarily include DNA polymerases, as well as topoisomerases, helicases, and ligases. There is growing evidence supporting the involvement of a highly organized multienzyme complex—the replisome—in DNA replication, which includes the primosome complex, helicases, Pol III holoenzyme, and gyrases.

DNA polymerases are the Key Enzymes of the replication process that actually drive the elongation of polynucleotide chains using the principle of complementarity. The DNA polymerases of the bacterium E. coli have been studied most thoroughly. Three Different types of DNA polymerases (Pol I, Pol II, and Pol III) have been discovered in these bacterial cells, differing primarily in catalytic speed and nuclease activity. DNA polymerase I (Pol I) is a single polypeptide containing about 1,000 amino acid residues. There are approximately 400 molecules of this enzyme in an E. coli cell. Pol I exhibits the following activities: polymerase activity—adding deoxynucleotides complementary to the template strand to the free 3'-OH group of a primer in the 5' to 3' (5' → 3') direction of the growing DNA molecule; exonuclease activity—hydrolyzing phosphodiester bonds (cleaving nucleotides) in a single DNA strand or at the unpaired end of a duplex DNA, starting from either the 3' end (3' → 5') or the 5' end (5' → 3'). Exonuclease activities play a crucial role in the Replication and Repair of E. coli chromosomal DNA. The 3' → 5' exonuclease activity ensures monitoring of the incorporation of each nucleotide and the removal of erroneous nucleotides from the growing end of the chain (proofreading), while the 5' → 3' exonuclease activity is used to remove pyrimidine dimers and ribonucleotides of Okazaki fragments.

DNA polymerase II (Pol II) is present in significantly fewer copies in Bacterial cells and carries out polymerase activity much slower than Pol I (accounting for only 5% of DNA polymerase I activity). Unlike Pol I, this enzyme lacks 5' → 3' exonuclease activity. The exact role of this polymerase in replication is not fully understood. It is believed that this enzyme is not essential for DNA replication but may substitute for certain Functions of Pol I when the latter is damaged.

DNA polymerase III (Pol III) is the principal enzyme responsible for the replication of E. coli chromosomal DNA. Each cell contains only 10–20 molecules of this enzyme, yet it operates approximately 60 times faster than DNA polymerase I. Additionally, Pol III has a higher affinity for the template and ensures greater copying efficiency. Like Pol II, this enzyme lacks 5' → 3' exonuclease activity. Therefore, the replication of the lagging strand requires the participation of Pol I to remove RNA primers at the 5' ends of Okazaki fragments.

A greater number of DNA polymerases have been identified in Eukaryotic cells, but their functions are less well understood.

The function of topoisomerases is to resolve mechanical and topological problems during the unwinding of The Double Helix at the Replication fork. These enzymes alter the degree of supercoiling and create a "swivel" that allows the replication fork to move continuously. Two MAIN TYPES OF topoisomerases have been identified in various organisms: Type I topoisomerases nick one of the two strands, allowing the end segment of the double helix to rotate around the intact strand before rejoining the ends of the cleaved strand. Type II topoisomerases make transient double-stranded breaks in both complementary strands, alter the supercoiling degree, and subsequently rejoin the broken ends.

Helicases drive the formation and progression of the replication fork—the region of the molecule with unwound strands—along the DNA helix. These enzymes utilize The energy released from ATP Hydrolysis to unwind the strands. To ensure a higher unwinding rate, several helicases act in a complex with single-stranded DNA-binding Proteins (SSBs) that bind to single-stranded Regions of the molecule, thereby stabilizing the unwound duplex.

Finally, DNA ligases catalyze the rejoining of DNA chain fragments by participating in The formation of covalent bonds (phosphodiester bridges) between the 5'-P and 3'-OH groups of adjacent deoxyribonucleotides. These enzymes also utilize the energy of high-energy bonds generated during the hydrolysis of ATP or GTP.

The Mechanism of double-stranded DNA replication has been best investigated in the bacterium E. coli and will be examined using this example.

Initiation of DNA replication. The replication of E. coli DNA begins at a strictly defined site called the origin (ori), or origin of replication, located at the 85th minute of the bacterial chromosome's genetic map. At the replication ori, enzymes (topoisomerases, helicases) act on the DNA to form a replication fork, where the actual copying of the strands takes place. Replication requires: a DNA template in the form of a single-stranded DNA region, a mixture of deoxynucleoside triphosphates, a replisome (the enzymatic assembly involved in replication), and a nucleic acid 3'-OH group acting as a primer to which the DNA polymerase attaches the next nucleotide. The fact is that none of the DNA polymerases can initiate nucleotide polymerization de novo. This function is performed by RNA polymerases, which recognize the replication ori within the replication fork and synthesize short (10–60 ribonucleotides) sequences known as RNA primers. Primer synthesis proceeds in the 5' to 3' direction, resulting in a free 3'-OH end that DNA polymerase can use to continue chain polymerization during the elongation stage of replication (Fig. 1.10).

Elongation of DNA replication. The synthesis of new DNA strands proceeds in accordance with the principle of complementarity: each nucleotide incorporated into the growing chain must be complementary to the corresponding (opposite) nucleotide in the parental (template) strand.

Because all DNA polymerases carry out nucleotide polymerization in only one direction (5' → 3'), while the replication fork moves along the DNA in both directions, only one strand—called the leading strand—can be synthesized continuously in each direction. The second (opposite) strand is synthesized in short segments (Okazaki fragments) and is called the lagging strand (Fig. 1.10). Okazaki fragments consist of approximately 1,000 nucleotides in prokaryotes and 100–200 nucleotides in eukaryotes.

In addition to chain polymerization, which is primarily carried out by DNA polymerase III, the following events occur during DNA replication:

— excision of RNA primers from the leading strand and each Okazaki fragment. This function is performed by Pol I utilizing its 5' → 3' exonuclease activity;

— Filling of the "gaps" left after the removal of RNA primers. This task is also performed by DNA polymerase I using the free 3'-OH group of the adjacent Okazaki fragment;

— joining of DNA fragments in the lagging strand with the help of DNA ligase: when the growing 3'-hydroxyl end of each Okazaki fragment reaches the 5'-deoxynucleotide end of the neighboring fragment, DNA ligase steps in to form a continuous lagging strand;

— correction of replication errors—proofreading. This mechanism is characteristic of both Pol I and Pol III and is based on their 3' → 5' exonuclease activity. DNA polymerase checks the complementarity of the incoming nucleotide by monitoring the size of the newly proposed nucleotide pair within its Active Site, and its polymerase activity is triggered only once this complementarity is verified. Furthermore, each newly incorporated nucleotide is also checked for proper pairing within the enzyme's active site. If the size of the formed nucleotide pair does not match the correct standard (i.e., when the opposing bases are not complementary), the enzyme excises the non-complementary nucleotide using its 3' → 5' exonuclease activity and seeks a replacement. An additional mechanism that reduces replication errors is DNA Repair. As a result, the frequency of erroneous nucleotide incorporation into the newly synthesized DNA chain is extremely low (10^-8–10^-10).

Fig. 1.10. Replication in E. coli. Arrows indicate the direction of new DNA strand synthesis. Unshaded thick lines represent RNA primers initiating synthesis; solid bold lines represent elongating DNA chain fragments. Vertical lines indicate Hydrogen Bonds between complementary nucleotides.

Termination of replication. In Bidirectional Replication of a circular genome (such as in Escherichia coli), the replication forks meet at a point 180° away from THE ORIGIN OF replication, where the process concludes. The circular DNA molecules are joined by a ligase at the meeting point, leaving them interlinked as catenanes; subsequently, they are resolved into separate genomes by type II topoisomerase.

The rate of DNA replication in E. coli is approximately 1,500 nucleotide pairs per second. Thus, the complete genome of the bacterium (4⋅10⁶ bp) is replicated in about 40 min. However, E. coli cells divide faster—every 20 min—which means that at the same rate of copying, the frequency of initiation events at the origin must increase. That is, before the first round of genome replication is completed at the ori site, a second round of replication is initiated. The rate of replication fork movement in eukaryotic cells is considerably lower (10–100 bp per second), but timely completion is ensured by simultaneous initiation at multiple origins. As a result, the Drosophila chromosome, for instance, containing 6.5⋅10⁷ bp, is replicated in just a few minutes.

Overall, the patterns of replication identified in prokaryotes are characteristic of most Eukaryotic Genomes as well. The differences lie primarily in the presence of multiple replication initiation sites on each chromosome in eukaryotes, alternative mechanisms for correcting replication errors, and the enzymatic machinery involved in the process. A schematic representation of replication processes in circular genomes (typical of prokaryotes and Plasmids) and linear genomes (eukaryotic) is shown in Fig. 1.11.

In linear DNA, strand unwinding occurs by the rotation of one strand around the other. In circular DNA, unwinding and replication lead to the formation of a structure resembling a ring with an internal loop. It is called a theta loop because its shape resembles the Greek letter Θ. Such loops can be observed in autoradiographs of replicating bacterial DNA, a technique first applied by Cairns to E. coli DNA.

The mechanism of bidirectional DNA replication described above is the most widespread, but not the only one. The DNA of phages P22, 186, P2, as well as phages T4 and $\lambda$, during the late Stages of the lytic cycle, replicates via a unidirectional mechanism (the rolling-circle type). In this case, double-stranded Circular DNA is nicked by a specific enzyme at a unique site in one strand (the rolling-circle origin). The resulting 5'-end of the strand binds to the enzyme that made the nick. DNA Synthesis begins with the Displacement of the enzyme-bound 5'-end into the solution, allowing DNA polymerase to add nucleotides to the 3'-OH end. This semiconservative replication proceeds with the 5'-end of the broken strand being displaced as a free tail of increasing length, while the intact closed strand serves as a template. This replicating structure (Fig. 1.12) is called a rolling circle, since the unwinding of the free single strand is accompanied by the Rotation of the double-stranded template around its axis.

If this mechanism is used to replicate double-stranded DNA, the 5'-terminal tails serve as templates for the synthesis of short DNA fragments, which are immediately joined together by DNA ligase. As a result, the growing tails acquire a double-stranded structure shortly after their formation. Elongation of the tails sometimes results in their length significantly exceeding the total length of the original circular molecule. This mode of replication is used, for example, by phage $\lambda$. During DNA packaging into capsids, nicks are introduced at special regions called cos-sites, spaced apart by the length of the viral genome. This cleaves the long duplexes of repeatedly replicated phage DNA into fragments corresponding in size to the mature DNA found in bacteriophage $\lambda$ virions.

Fig. 1.11. Types of bidirectional replication of double-stranded DNA. Solid lines indicate original DNA strands; dashed lines represent newly synthesized strands.

Rolling-circle replication is also characteristic of the formation of copies of the bacterial chromosome of E. coli Hfr and the F⁺ factor, which are transferred to the recipient cell during conjugation.

Replication of single-stranded DNA. In phages M13 or $\phi$X174, whose mature genomes are represented by single-stranded circular DNA molecules, replication proceeds via the rolling-circle mechanism (Fig. 1.12). This occurs during the late stages of infection, after the infecting DNA has been converted into a double-stranded circular form. Unlike the replication of phage $\lambda$ genomes (Fig. 1.12, pos. 5), the 5'-terminal regions are not replicated in this case; therefore, the products of replication are long single DNA strands that continuously peel off from the "rolling spool." These strands are cleaved at each replication origin and circularize to form mature forms packaged into capsids.

Fig. 1.12. Rolling-circle replication of double-stranded DNA. Solid lines indicate original DNA strands; dashed lines represent strands newly synthesized during replication. Position 1 — original circular DNA; 2 — nick formation in one of the strands exposing the 3'- and 5'-ends; 3, 4 — replication of the circular strand at the replication fork; 5 — initiation of 5'-tail replication; 6, 7 — replication products.

RNA replication. Introduction/7.html">RNA-containing Viruses reproduce via the replication of their RNA, whereas all cellular RNAs are synthesized via DNA Transcription. With the exception of Retroviruses, RNA replication largely mirrors DNA replication. As in DNA replication, The nucleotide sequence is determined by complementary copying of a template, which in this case is necessarily an RNA strand. The enzymes catalyzing this process are called RNA-dependent RNA polymerases (replicases). The RNAs of bacterial viruses R17 and MS2, as well as animal-infecting polioviruses and Sindbis virus, are always designated as plus-strand (+) because their RNA genome sequence is identical to that of mRNA. Thus, the infecting viral genome can serve as mRNA and carries the information for the synthesis of some, if not all, viral proteins. A specific replicase encoded by the viral genome, synthesized shortly after infection, binds to one or more host cell proteins and initiates the copying of the (+)-strand from its 3'-end, yielding a full-length (-)-strand associated with the (+)-strand template. Subsequently, the same replicase synthesizes numerous copies of the (+)-RNA strand using the newly synthesized (-)-strand as a template. The genomes of certain viruses (vesicular stomatitis virus, Influenza virus) consist of one or more (-)-strands. In this case, they serve as templates for the synthesis of (+)-strands, which function as mRNAs and are used to synthesize progeny (-)-strands.

A distinguishing feature of retroviral genome replication is that upon penetration of their RNA into the host cell, the viral genome undergoes reverse transcription. This process first yields an RNA-DNA duplex and subsequently a double-stranded DNA molecule. The enzyme that catalyzes the complementary copying of RNA into DNA is called Reverse Transcriptase (revertase). It is contained within retroviral particles (virions) and becomes activated upon entry into The Cell. Accumulating evidence indicates that reverse transcription occurs in A wide variety of eukaryotic cells, and reverse transcriptase plays a vital role in genome rearrangement processes. Replication of the double-stranded retroviral DNA form does not commence until it integrates into the host cell DNA. The mechanism of recombinational integration is not yet fully understood. Once integrated, the retroviral DNA replicates as a part of the cellular DNA. The RNA of progeny virions is produced by the Transcription of the integrated copies of the viral DNA.