BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 10. NUCLEIC ACID METABOLISM

10.6. DNA Biosynthesis (Replication)

10.6.2. Mechanism of DNA Replication

DNA Biosynthesis consists of A large number of sequential stages, including recognition of the Replication origin, unwinding of the parental double helix, Formation of the Replication fork (initiation), synthesis of new strands (elongation), and completion of the synthesis of two daughter DNA strands (termination).

Replication initiation. Since DNA Synthesis occurs on a single-stranded template, it must be preceded by the obligatory Separation of the two DNA strands. The process begins at a specific site (the origin) simultaneously in both directions through The formation of replication forks. At these replication sites, specialized Enzymes called DNA topoisomerases act on the DNA, introducing single- or double-strand breaks followed by the unwinding of supercoils and

subsequent restoration of strand continuity. The joining of the broken strand occurs without additional energy consumption, as the required Energy is stored in the form of a high-energy (macroergic) covalent bond formed between the pentose-phosphate backbone of the DNA strand and the topoisomerase. Through this action of DNA topoisomerases, the separation of the two strands at the replication fork is facilitated. The breaking of Hydrogen Bonds, the divergence of polynucleotide strands, and the progression of the replication fork are driven by DNA helicase enzymes, which require ATP Hydrolysis energy for their function. Next, special helix-destabilizing Proteins, known as SSB proteins, bind to the single-stranded DNA regions, preventing the individual DNA strands from re-annealing. This selective affinity of SSB proteins for single-stranded DNA regions is due to the presence of hydrophobic pockets where nitrogenous bases can insert. Furthermore, SSB proteins interact cooperatively with one another, aligning in long rows along the single-stranded DNA and thereby stabilizing it in this state.

The entire complex of proteins involved in the replication process forms a complex supramolecular Structure at the METABOLISM/36.html">DNA replication fork known as the replisome.

Since DNA polymerases are incapable of initiating chain synthesis on their own, another enzyme is required to provide a primer polynucleotide chain. This enzyme, DNA primase, does not require a free 3'-OH group for initiation, copies a portion of the DNA template strand in the 5' to 3' direction, and thereby generates a short (10-60 ribonucleotides) single-stranded RNA molecule (RNA primer) with a free 3'-OH end. Subsequently, DNA primase dissociates, and DNA polymerase utilizes the 3'-OH group of the primer to continue the polymerization of the DNA strand during the elongation stage of replication.

Replication elongation. The synthesis of new DNA strands is carried out according to THE PRINCIPLE OF complementarity: each nucleotide incorporated into the growing (daughter) strand must be complementary to the corresponding nucleotide in the template (parental) strand.

DNA polymerases catalyze nucleotide polymerization exclusively in the 5'→3' direction, while the enzyme moves along the template in the 3'→5' direction. Consequently, the continuous elongation of the growing strand can proceed along only one template strand—specifically, the one relative to which the replication fork moves from the 3' to the 5' end. The strand synthesized continuously is called the leading strand (Fig. 10.17).

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Fig. 10.17. Scheme of DNA replication

On the second template strand, the Synthesis of the new strand proceeds in the 5'→3' direction, but opposite to the movement of the replication fork. Therefore, the second strand is synthesized discontinuously, in short fragments known as Okazaki fragments (named after the researcher who discovered them), and is referred to as the lagging strand.

Each Okazaki fragment begins with a corresponding RNA primer and ends just before THE START OF the preceding primer. In prokaryotes, the length of an Okazaki fragment is about 1,000 NUCLEOTIDES, whereas in eukaryotes it is 100-200 nucleotides. This difference in Okazaki fragment length and DNA synthesis speed is explained as follows. Unlike in prokaryotes, DNA replication in eukaryotes does not occur in a naked state, but rather within Chromatin. As is well known, chromatin consists of nucleosomes, each wrapping around 200 Base Pairs of DNA. This is likely the reason why Okazaki fragments of the eukaryotic lagging strand are 100-200 nucleotides long. According to modern concepts, nucleosomes create a barrier capable of impeding the movement of DNA polymerase. Consequently, it becomes clear why The rate of replication in eukaryotes is significantly lower than in prokaryotes.

Prokaryotic DNA polymerase III (eukaryotic DNA polymerase δ) successively extends the chain by adding corresponding deoxyribonucleotides one by one (Fig. 10.18). Through one site of its active center, the enzyme captures the first unpaired template nucleotide in the replication fork zone; through the second site, it binds the growing end of the polynucleotide chain containing a free OH group at the 3'-end; and through the third site, it binds one of the free deoxyribonucleoside triphosphates that is complementary to the unpaired template nucleotide located in the first Active Site. In this state, the enzyme attacks the α-phosphodiester bond of the bound nucleoside phosphate, releasing pyrophosphate, while the resulting nucleoside monophosphate is attached via a 3',5'-phosphodiester bond to the 3'-OH group of the growing chain.

Fig. 10.18. Elongation of DNA replication

The utilization of dNTPs renders the formation of phosphodiester bonds thermodynamically favorable, since phosphodiester formation from a monophosphate and an OH group is coupled with the hydrolysis of the anhydride pyrophosphate bond.

In addition to chain polymerization during DNA replication, RNA primers are excised from the leading strand and from each Okazaki fragment. In prokaryotes, this function is performed by DNA polymerase I (a similar function in eukaryotes is carried out by DNA polymerase β). The enzyme progressively removes ribonucleotides from the 5'-end of the primer one by one using its 5'→3' exonuclease activity. Simultaneously, DNA polymerase adds deoxyribonucleotides to the OH group at the 3'-end of the preceding Okazaki fragment in an amount equal to the excised primer, thereby filling the gap created by the removal of the ribonucleotides.

The joining of DNA fragments on the lagging strand is mediated by the enzyme DNA ligase, which catalyzes the formation of a phosphodiester bond between the 5'-phosphate of one strand fragment and the 3'-OH group of the deoxyribose of the adjacent fragment, resulting in a continuous DNA strand.

Unlike the polymerase reaction involving DNA polymerase, where one of the participants is a free deoxyribonucleoside triphosphate, in the DNA ligase reaction both participants—the terminal deoxyribonucleoside monophosphates—are constituents of the joining fragments. Furthermore, DNA ligases only join single-stranded fragments that are part of a double-stranded DNA molecule.

Replication termination. The termination of DNA replication depends on The structure of the DNA. During the replication of circular DNA, the continuous growth of the leading and lagging strands along the template inevitably leads to the Displacement of the 3'-OH and 5'-phosphoryl ends of a single strand at THE ORIGIN OF replication. In Bidirectional Replication of circular DNA, when two replication forks moving in opposite directions are formed, DNA synthesis terminates at a distance of 180° from the origin. At the points of convergence, circular DNA molecules are joined by DNA ligase, leaving them catenated (linked in pairs). Subsequently, they are resolved into separate genomes by type II topoisomerases.

In the case of linear DNA molecules, termination and completion of replication occur by dividing The Genome into numerous individual replicons. Such replicons are not all activated simultaneously, but (as in eukaryotes) Cell Division must be preceded by an obligatory single round of replication for each of them. The progression of the replication fork halts only upon collision with another fork moving in the opposite direction or upon reaching the end of the chromosome. As a result, the entire chromosomal DNA is replicated within a short period of time.

Upon completion of replication, two DNA chains are formed that are identical to the original strands. Each parental strand remains intact and serves as a template for the synthesis of a complementary strand. The resulting new double-stranded molecule thus contains one original strand and one newly synthesized strand.

Replication of DNA telomeric regions. The terminal regions of eukaryotic Chromosomes—telomeres—contain specific nucleotide sequences represented by numerous tandemly arranged oligonucleotide sequences (hexanucleotides - TTAGGG). Such sequences are referred to as telomeric sequences (telomeric DNA). The total length of these repeats at one end of the DNA at the initial stage of an Organism's ontogenetic development ranges from 10,000 to 15,000 nucleotide pairs.

The presence of telomeric repeats is essential for completing the replication of the terminal informative sequences in chromosomal DNA, thereby preserving Genetic information.

Upon completion of linear DNA molecule replication, the 5' ends of its daughter strands remain unreplicated because RNA primers located at these ends are removed (Fig. 10.19).

It is important to note that in the case of circular bacterial DNA replication, this problem does not arise, as the initially formed RNA primers are removed by an enzyme that simultaneously fills the resulting gap by extending the 3'-OH end of the growing DNA strand.

Telomeric repeats carry no genetic information; therefore, the loss of a portion of these sequences does not disrupt genome function—by their very existence, they protect more vital DNA sequences from incomplete replication.

With each cell division, chromosomal DNA shortens by approximately 0.00005% of its length. At first glance, this is a negligible value that could seemingly be ignored. However, considering the sheer number of cell divisions, the absence of mechanisms to restore the length of chromosomal telomeric DNA can lead to catastrophic consequences.

Fig. 10.19. Shortening of newly synthesized DNA strands after the removal of RNA primers

Terminal under-replication of chromosomal DNA is of great biological significance. According to current models, telomere shortening to a critical level triggers somatic cell senescence, and upon reaching this threshold, leads to cell death (the telomere theory of Aging).

However, in Germ Cells, stem cells, and other rapidly dividing cells, a mechanism exists to lengthen these unreplicated DNA regions. Eukaryotic cells possess an enzyme called terminal deoxynucleotidyl transferase (telomerase), which ensures the restoration of unreplicated 5' ends. As a prosthetic group, telomerase contains an RNA molecule approximately 450 nucleotide pairs long. It simultaneously serves as a template for synthesizing telomeric repeats and binds via Hybridization to the terminal telomeric region of DNA. Using this RNA, telomerase extends not the daughter strand, but the parental strand in the 5'→3' direction based on the principle of complementarity (Fig. 10.20).

Fig. 10.20. Elongation of the DNA telomeric region involving telomerase

To the 3' end of the DNA strand, telomerase successively attaches the TTAGGG hexanucleotide. It then shifts along the DNA strand by one telomere and initiates the synthesis of a new hexanucleotide. Chain extension via telomerase continues until the elongated strand is capable of acting as a template for the formation of another Okazaki fragment of the new strand.

First, primase synthesizes An RNA primer on the daughter strand, after which DNA polymerase β sequentially attaches deoxyribonucleotides complementary to the parental strand's telomeric repeats to the primer. Replication of the fragment proceeds in the 5'→3' direction and ceases upon encountering the preceding 5' end of the daughter strand. Ligation of the fragment to the strand is carried out by DNA ligase, while an exonuclease removes the RNA primer from the 5' end of the extended daughter strand.

Thus, the end of the double-stranded DNA acquires the original conformation it had prior to telomerase action; although the daughter strand remains shorter than the parental strand, it is effectively longer by a series of telomeric repeats.

In most somatic Cells of the organism, the telomere replication mechanism is absent—telomerase is inactive, and consequently, telomeric DNA progressively shortens with each cell division. Such cells possess a length of telomeric DNA sufficient for the lifespan of The Cell and its progeny. Nevertheless, low telomerase activity is known to be detectable in cells with a high renewal rate (lymphocytes, epidermal Skin cells, epithelial cells, etc.).