Fundamentals of Biochemistry - A. A. Anisimov 1986

Nucleic Acids
DNA Synthesis

4.7.1. Semiconservative METABOLISM/36.html">DNA Replication. J. Watson and F. Crick not only developed a Introduction/20.html">DNA Structure model consistent with all experimental data but also proposed a hypothesis for The Mechanism of DNA synthesis via doubling (replication). According to this mechanism, a double-stranded DNA molecule first splits lengthwise, and its two strands separate. A new strand is formed on each old strand. NUCLEOTIDES of the new strands pair complementarily with the nucleotides of the old strands, so that the old strands serve as templates. This produces two daughter double-stranded DNA molecules that are completely identical to the parental molecule. In each daughter molecule, one strand is inherited from the parental DNA, while the second is newly synthesized. This pathway of synthesis was named semiconservative replication.

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Fig. 4.14. Possible modes of DNA reproduction:

I — conservative, II — semiconservative, III — dispersive

However, theoretically two other Mechanisms for the equal distribution of DNA between daughter Cells are possible, known as conservative and dispersive replication. In conservative replication, a new DNA molecule is synthesized on the two strands of the parental DNA molecule without their Separation. The dispersive mechanism involves the fragmentation of DNA molecules, As a result of which each individual strand of the new daughter molecules contains regions of both the old and the new DNA strand (Fig. 4.14).

M. Meselson and F. Stahl (1958) proved in experiments with E. coli that DNA replication in The Cell occurs in a semiconservative manner. They grew E. coli on a synthetic medium containing glucose and salts. The nitrogen source NH₄Cl contained heavy ¹⁵N instead of the usual light ¹⁴N, so that all nitrogen-containing substances of the Bacteria, including DNA, became "heavy." The bacterial cells were then washed and transferred to a "light" nutrient medium containing the nitrogen isotope ¹⁴N, where further growth took place. At various intervals, DNA was extracted from the bacteria and centrifuged in a CsCl density gradient. For the zero generation, a single band corresponding to ¹⁵N-DNA was obtained. After brief growth, during which the cell number doubled, another, lighter band appeared in the gradient. Its density corresponded to a hybrid DNA molecule containing equal amounts of ¹⁵N and ¹⁴N. Apparently, the strands with ¹⁴N were newly synthesized. This is precisely the result expected based on the semiconservative mechanism. Following DNA Replication and the completion of two bacterial division cycles, two DNA bands of different densities were detected in the density gradient. One of these bands corresponded to hybrid DNA containing ¹⁵N and ¹⁴N, and the second to DNA containing both strands with ¹⁴N. These data convincingly prove that DNA synthesis proceeds in a semiconservative manner.

4.7.2. DNA Replication as a Multi-Step Process. For DNA to replicate in a semiconservative manner, it must separate into its constituent strands. It has been established that DNA strands do not unwind along their entire length, but rather at a short segment. Here, a Replication fork is formed—the site of DNA duplication. Movement of the replication fork is possible only by unwinding the double-helical DNA and winding the two daughter molecules. Helix conformation is altered by A number of Enzymes, which are divided into three groups.

The first group comprises helicases—Proteins that unwind The Double Helix and prevent single strands from re-associating. In E. coli, these include the single-stranded DNA-binding protein and the rep protein.

The second group consists of swivelases (topoisomerases I, or relaxing proteins). They help eliminate DNA Supercoiling (i.e., they function as a swivel). Swivelases introduce a break into one of the DNA strands, making it possible for this strand to unwind, followed by resealing of the break. In E. coli, the ω-protein belongs to this group.

The third group comprises gyrases—topoisomerases II. They induce negative supercoiling in circular DNA by breaking a single bond, rotating the strand, and resealing the bond. In effect, they also act as a swivel.

At the E. coli replication fork, the rep protein destabilizes the DNA double helix, while the binding protein attaches to single-stranded regions, enabling them to serve as templates. Gyrases apparently facilitate The formation of left-handed, i.e., (—) supercoils, which creates (—) superhelical pressure on the fork and eases the work of helicases.

Phage and bacterial genomes replicate as a single entity. They represent organized units of replication, the so-called replicons. A replicon has an origin—ori (from origin), a directed orientation, and sometimes a terminal region—ter.

Replication begins at the ori region. Here, the DNA strands separate, forming two replication forks in which new DNA strands are synthesized. If the DNA is circular, the replication forks move toward each other until the DNA is completely copied (or until the ter point). Unidirectional replication has been discovered in some Plasmids, in which a single replication fork moves in a defined direction.

The DNA replication process is divided into three main stages: 1) initiation, 2) elongation, or strand growth, and 3) termination. According to electron microscopic, autoradiographic, and genetic data, the synthesis of two oppositely oriented DNA strands occurs at high speed within the replication fork. The direction of one strand, 5'→3', coincides with the direction of fork movement; this strand is called the leading strand. The second strand is called the lagging strand. In 1968, R. Okazaki and coworkers showed that DNA synthesis occurs on both template DNA strands in the 5'→3' direction and proceeds discontinuously, in separate fragments 1–2 thousand nucleotides in size. These fragments were subsequently named Okazaki fragments. Larger fragments are formed in the leading strand of DNA, and the direction of their growth (5'→3') coincides with the Direction of replication fork movement. Over time, Okazaki fragments increase in size and ultimately form continuous daughter DNA strands. It is even suggested that in E. coli the leading strand grows continuously. In the lagging strand of DNA, short fragments are formed, which is why synthesis initiation occurs repeatedly.

DNA synthesis is carried out by DNA polymerases, for which RNA fragments serve as primers in vivo.

Protein B plays an important role in the initiation of replication at the ori site. After unwinding of the double helix, the B-protein binds to the leading strand template at the ori site; it initiates its primer, then moves in the opposite direction and ensures the initiation of the lagging strand primers. Thus, the ß-protein acts in E. coli as a "mobile promoter," i.e., a Transcription initiation site.

Primer synthesis is carried out by primases (specialized RNA polymerases). The 3'-end of the RNA primer is used by the primase to initiate a DNA fragment, and its subsequent elongation is performed by the DNA polymerase III holoenzyme. Therefore, each Okazaki fragment begins with an RNA fragment of 50–200 nucleotides, which is subsequently removed. Removal of primers from Okazaki fragments in E. coli is performed by DNA polymerase I, which also fills the resulting gaps. The enlargement of Okazaki fragments by their ligation occurs with the participation of DNA ligase (Fig. 4.15).

The mechanisms of replication termination are insufficiently understood. A specialized region—the terminator—exists for termination in E. coli DNA. Terminators are absent in the genomes of a number of Bacteriophages; Bidirectional Replication presumably concludes after the meeting of two forks and replicating complexes.

The details of Eukaryotic Chromosome replication are not fully understood, but the Selection/11.html">General features of the process can be outlined as follows. A eukaryotic chromosome is a polyreplicon structure, meaning it contains numerous independent replicons, each containing an origin and a terminator. Adjacent replicons are oppositely oriented; upon completion of replication, their replicas—that is, the synthesized complementary strands—fuse. Replicon sizes range from 10 to 100 µm, which corresponds to 3×10⁴–3×10⁵ nucleotide pairs. High replication speed is ensured by the formation of A large number of replication forks.

Fig. 4.15. E. coli DNA replication fork (showing replication stages and participating proteins)

Semiconservative replication is initiated at the ori site by nicking one of the strands of the supercoiled molecule and, consequently, separating the DNA strands. A replication fork is formed here. Continuous synthesis proceeds along the leading DNA strand in the fork, and discrete synthesis along the lagging strand. Okazaki fragments range from 40 to 290 nucleotides in size (averaging 135), and primers are about 9 nucleotides long. The replication mechanism is apparently similar to that of prokaryotes. DNA synthesis is carried out by DNA polymerase α. Other cellular enzymes performing Functions similar to those of prokaryotic replication enzymes have also been isolated.

Features of DNA replication in Chromosomes are related to the latter's nucleosomal Organization. When the replication fork passes through a nucleosomal DNA region, this region is "unwound" with the help of helix-destabilizing enzymes. After passage of the fork, two new Double helices are formed, and the nucleosomal structure is restored.

Replication of viral and phage genomes is frequently accomplished using host cell enzymes. However, it may be organized differently than in bacteria.

Oligomeric plasmid genomes are frequently detected in plasmid-infected bacteria. Several identical replicons of a single plasmid turn out to be joined within a single DNA molecule. The formation of such forms is explained by a DNA replication model known as the "rolling circle" (Fig. 4.16). According to this model, one strand of a double-stranded circular DNA molecule is broken, and its 5'-end attaches to The Cell wall membrane. Semiconservative replication then takes place, during which the second strand retains its circular form: it acts as if "rolling," serving as an endless template. If replication terminates at the exact point where it began, a monomer genome is synthesized; if the circle "rolls" further, oligomers containing multiple genomes will appear. Such replication products can monomerize and acquire a circular form via recombination. It is hypothesized that rolling-circle replication occurs in a number of E. coli bacteriophages, such as P2, P22, T4, λ, and during bacterial conjugation. In Eukaryotic cells, this mechanism drives Amplification, i.e., selective multi-round Gene replication. An example is the amplification of genes encoding rRNA in Xenopus oocytes.

A. Kornberg (1977) suggests that enzymes involved in DNA synthesis are organized into a complex. They form particles 8–12 nm in diameter, attached to The cell membrane via hydrophobic interactions. This complex is called the replisome.

Once the DNA strands are formed, DNA methylases methylate them, conferring species Specificity upon the DNA. DNA gyrase catalyzes the formation of supercoils.

4.7.3. DNA Damage Repair. Environmental chemical and physical factors can cause DNA damage. For instance, high doses of ultraviolet radiation are lethal, whereas low doses exhibit antimitotic and mutagenic effects. This induces several alterations in the DNA molecule: cytosine undergoes deamination, turning into uracil, which can trigger Mutations; thymine bases form dimers, which are double thymine rings; and regions of local DNA Denaturation appear, hindering DNA replication.

Fig. 4.16. Rolling-circle mechanism of DNA replication (after G. Meynell, 1976);

1 — original double-stranded molecule, 2 — newly synthesized strands, 3 — 3'-end, 4 — 5'-end

Throughout evolution, the cells of living organisms have developed mechanisms for DNA damage repair. One such repair process occurs in the presence of light and is known as photoreactivation. An enzyme associated with a chromophore is known to absorb visible light, providing the energy required to drive the reaction. The enzyme specifically binds to the thymine dimer, cleaves it, and then dissociates from the DNA. DNA functions are restored by approximately 90%.

Another well-studied process is dark (excision) DNA Repair, which involves the removal of the damaged segment. A specific endonuclease recognizes the lesion and nicks the DNA strand near the thymine dimer. Another enzyme, UV endonuclease, excises the oligonucleotide containing the thymine dimer. DNA polymerase I (or DNA polymerase II in other cases) fills the resulting gap by incorporating nucleotides. The enzyme DNA ligase seals the phosphodiester bond between the newly synthesized fragment and the rest of the DNA. Dark repair can correct numerous potentially lethal genome lesions. For example, in bacteria, it can repair polynucleotide strand breaks caused by X-rays. Dark repair can also remove purine base cross-links in DNA induced by mustard gas. Thus, repair systems enhance the stability of DNA as the carrier of hereditary information.

Certain rare hereditary human diseases are associated with defects in DNA repair pathways, xeroderma pigmentosum being one of them. Patients with xeroderma pigmentosum are hypersensitive to sunlight and are highly prone to Skin Cancer. When skin cell cultures from such patients are exposed to ultraviolet radiation, The rate of thymine dimer excision in these cells is lower than that in normal human skin cells. In one group of xeroderma pigmentosum patients, UV endonuclease activity is absent. In another group, cells are unable to repair single-strand DNA breaks, presumably due to a deficiency in DNA polymerase I.