DNA REPLICATION: MECHANISMS, IN PROKARYOTES AND EUKARYOTES - BIOLOGY - 2025

The replication of DNA (deoxyribonucleic acid) is to copy the genome, ie, all the genetic information in the DNA of an organism to produce two identical copies. The genome has the information necessary to build a complete organism.

Before cell division, DNA replication occurs. Through meiosis, gametes are produced for sexual reproduction. Through mitosis, cell replacement (eg, skin and blood) and development (eg, tissues and organs) occur.

Source: I, Madprime

Knowing the structure of DNA allows us to understand how its replication occurs. The structure of DNA consists of a double helix, composed of two antiparallel chains of successive nucleotides, whose nitrogenous bases complement each other in a specific way.

During replication, each strand of the DNA double strand acts as a template for the biosynthesis of a new strand. The two newly synthesized chains have bases that are complementary to the bases of the template chain: adenine (A) with thymine (T), and cytosine (C) with guanine (G).

Various enzymes and proteins are involved in DNA replication. For example, opening the DNA double helix, keeping the DNA open, and adding deoxyribonucleosides-5′-triphosphate (dNTP) to form the new strand.

DNA replication is semi-conservative

Based on the structure of DNA, Watson and Crick proposed that DNA replication occurs semi-conservatively. This was demonstrated by Meselson and Stahl by labeling the DNA of Escherichia coli with the heavy isotope of nitrogen, ¹⁵ N, following the distribution pattern in a culture medium with light nitrogen, ¹⁴ N for several generations.

Meselson and Stahl found that, in the first generation, the two daughter DNA molecules had each molecule labeled with a chain with the heavy isotope of nitrogen and another with the light isotope. Unlike the parent DNA molecule, which had both strands labeled with the heavy isotope, ¹⁵ N.

In the second generation, 50% of the DNA molecules were like those of the first generation, and the other 50% had only light nitrogen. The interpretation of this result is that the daughter double helix has a parent chain (which functions as a template) and a new chain.

The semi-conservative replication mechanism involves the separation of DNA strands and complementary base pairing through successive nucleotide pairing, producing two daughter double helices.

Battery replication

Initiation of DNA replication in bacteria

Bacterial DNA consists of a circular chromosome and has only one site of origin of replication. From this site, the biosynthesis of the two daughter chains occurs bidirectionally, forming two replication forks that move in opposite directions to the origin. In the end, the hairpins meet, completing the replication.

Replication begins with the binding of DnaA proteins to the site of origin. These proteins in turn form a complex. Then the HU and IHF proteins, among others, join together, which together bend the DNA, causing the separation of the two DNA strands in a region rich in thymine and adenine.

Next, DNaC proteins bind, which cause the DNA helicases to bind. They help unwind DNA and break hydrogen bonds, formed between base pairs. So the two chains are further separated, forming two simple chains.

Topoisomerase II, or DNA gyrase, moves in front of DNA helicase, decreasing positive supercoils. Single-stranded DNA-binding (SSB) proteins keep DNA strands apart. Thus, the biosynthesis of the daughter chain can begin.

Biosynthesis of daughter DNA strands in bacteria

The primase enzyme is responsible for synthesizing short RNA chains called primers, which are 10-15 nucleotides long. DNA polymerase begins to add 5′-triphosphate deoxynucleosides (dNTPs) to the 3′-OH end of the primer sugar, after which the strand continues to grow from the same end.

Because DNA strands are antiparallel, one primer is synthesized on the leader strand and many primers on the lag strand. Because of this, the biosynthesis of the delayed chain is discontinuous. Although the DNA strands are antiparallel, the replication fork moves in only one direction.

DNA polymerase is responsible for the formation of covalent bonds between adjacent nucleotides of the newly synthesized chains, in the 5'®3 ′ direction. In E. coli, there are five DNA polymerases: DNA polymerases I and III carry out DNA replication; and DNA polymerases II, IV and V are responsible for repairing and replicating damaged DNA.

Most of the replication is performed by DNA polymerase III, which is a holoenzyme that has 10 different subunits with various functions in DNA replication. For example, the alpha subunit is responsible for making links between nucleotides.

A complex of enzymes is responsible for the replication of DNA in bacteria

DNA helicase and primase join to form a complex called a primosome. This moves along the DNA, acting in a coordinated way to separate the two parental strands, synthesizing the primers every certain interval on the delayed strand.

The primosome physically binds to DNA polymerase III, and forms the replisome. Two DNA polymerases III are responsible for replicating the DNA of the guide and delayed chains. With respect to DNA polymerase III, the delayed strand forms an outward loop, which allows the addition of nucleotides to this strand to occur in the same direction as the leader strand.

The addition of nucleotides to the leader chain is continuous. While in the delayed it is discontinuous. Fragments 150 nucleotides in length are formed, called Okazaki fragments.

The 5 ′ -> 3 ′ exonuclease activity of DNA polymerase I is responsible for eliminating the primers and filling, adding nucleotides. A ligase enzyme seals the gaps between fragments. Replication ends when the two replication hooks meet in a termination sequence.

The Tus protein binds to the termination sequence, stopping the movement of the replication fork. Topoisomerase II allows the separation of the two chromosomes.

Deoxyribonucleotide triphosphates are used by DNA polymerase

Deoxynucleoside triphosphate (dNTP) contains three phosphate groups attached to the 5 ′ carbon of deoxyribose. The dNTPs (dATP, dTTP, dGTP and dCTP) bind to the template chain following the AT / GC rule.

DNA polymerase catalyzes the following reaction: The 3 ′ hydroxyl group (–OH) of the growing strand nucleotide reacts with the alpha phosphate of the incoming dNTP, releasing inorganic pyrophosphate (PPi). Hydrolysis of PPi produces the energy for the formation of the covalent bond, or phosphodiester bond, between nucleotides of the growing chain.

Mechanisms that ensure the fidelity of DNA replication

During DNA replication, DNA polymerase III makes a mistake by 100 million nucleotides. Although the probability of error is very low, there are mechanisms that ensure fidelity in DNA replication. These mechanisms are:

1) Stability in base pairing. The hydrogen bonding energy between AT / GC is higher than in wrong base pairs.

2) Structure of the active site of DNA polymerase. DNA polymerase preferentially catalyzes nucleotide junctions with correct bases on the opposite strand. A bad base pairing causes a distortion of the DNA double helix, which prevents the wrong nucleotide from occupying the active site of the enzyme.

3) Reading test. DNA polymerase identifies incorporated erroneous nucleotides and removes them from the daughter strand. The exonuclease activity of DNA polymerase breaks the phosphodiester bonds between nucleotides at the 3 ′ end of the new strand.

DNA replication in eukaryotes

Unlike replication in prokaryotes, where replication begins at a single site, replication in eukaryotes begins at multiple sites of origin and the replication fork moves bidirectionally. Subsequently, all the replication hairpins fuse, forming two sister chromatids joined at the centromere.

Eukaryotes possess many types of DNA polymerase, whose names use Greek letters. DNA polymerase α forms a complex with primase. This complex synthesizes short primers consisting of 10 nucleotides of RNA followed by 20 to 30 nucleotides of DNA.

Next, ε or δ DNA polymerase catalyzes elongation of the daughter strand from the primer. DNA polymerase ε is involved in the synthesis of the leader chain, while DNA polymerase δ synthesizes the retarded chain.

DNA polymerase δ elongates the Okazaki fragment on the left until it reaches the RNA primer on the right, producing a short flap of the primer. Unlike prokaryotes, where a DNA polymerase removes the primer, in eukaryotes a Flap endonuclease enzyme removes the RNA primer.

Next, a DNA ligase seals the adjacent DNA fragments. Completion of replication occurs with the dissociation of proteins from the replication fork.

The replication of DNA in eukaryotic cell cycle and

Replication in eukaryotes occurs in the S phase of the cell cycle. The replicated DNA molecules are secreted into two daughter cells during mitosis. The G1 and G2 phases separate the S phase and mitosis. Progression through each phase of the cell cycle is highly regulated by kinases, phosphatases, and proteases.

In the G1 phase of the cell cycle, the origin recognition complex (OCR) binds to the site of origin. This induces the binding of MCM helicases and other proteins, such as Cdc6 and Cdt1, to form a pre-replication complex (preRC). The MCM helicase binds to the guide chain.

In S phase, preRC becomes an active replication site. The OCR, Cdc6, and Cdt1 proteins are released, and MCM helicase moves in the 3 ′ to 5 ′ direction. Once replication is finished, it will be restarted in the next cell cycle.

Replication of the ends of chromosomes in eukaryotes

The ends of chromosomes are known as telomeres, which consist of repeated tandem sequences, and a 3 ′ region that protrudes, 12 to 16 nucleotides in length.

DNA polymerase is unable to replicate the 3 ′ end of DNA strands. This is due to the fact that DNA polymerase can only synthesize DNA in the 5'-3 'direction, and can only lengthen pre-existing strands, without being able to synthesize a primer in this region. Consequently, telomeres shorten with each round of replication.

The enzyme telomerase prevents the shortening of telomeres. Telomerase is an enzyme that possesses protein and RNA subunits (TERC). The latter binds to the repeating sequences of DNA, and allows telomerase to bind to the 3 ′ end of the telomere.

An RNA sequence behind the junction site functions as a template for the synthesis of a six nucleotide sequence (polymerization) at the end of the DNA strand. Telomere elongation is catalyzed by subunits of telomerase, termed telomerase reverse transcriptase (TERT).

After polymerization, translocation takes place, consisting of the movement of telomerase to a new end of the DNA chain, joining another six nucleotides until the end.

The functions of other DNA polymerases in eukaryotes

DNA polymerase β plays an important role in removing incorrect bases from DNA, but it is not involved in DNA replication.

Many discovered DNA polymerases belong to the group of "translesion-replicating" polymerases. These polymerases are responsible for synthesizing complementary strands in a region of damaged DNA.

There are several types of "translesion-replicating" polymerases. For example, DNA polymerase η can replicate on thymine dimers, which are produced by UV light.

DNA replication in archaebacteria

Archaebacterial DNA replication is similar to that in eukaryotes. This is due to the following: 1) the proteins that participate in replication are more similar to those of eukaryotes than those of prokaryotes; and 2) although there is only one replication site such as in prokaryotes, its sequence is similar to the site of origin of eukaryotes.

The similarity in replication between the Archea and the eukaryotes supports the idea that both groups are phylogenetically more related to each other than either to prokaryotes.

References

1. Brooker, RJ 2018. Genetics analysis and principles. McGraw-Hill, New York.
2. Hartwell, LH, Goldberg, ML, Fischer, JA, Hood, L. 2018. Genetics - from genes to genomes. McGraw-Hill, New York.
3. Kušić-Tišma, J. 2011. Fundamental aspects of DNA replication. InTech Open access, Croatia.
4. Lewis, R., 2015. Human genetics concepts and applications. McGraw-Hill, New York.
5. Pierce, BA 2005. Genetics - a conceptual approach. WH Freeman, New York.