DNA replication: models, enzymes and stages of the process

Models of DNA replication

Replication is the process of forming two daughter DNA molecules from a single parent molecule. There are three theoretical models: semi-conservative, conservative, and dispersive.

• In the semi-conservative model, each daughter molecule consists of one parental and one newly synthesized polynucleotide chain.
• In the conservative model, the original double-stranded molecule is conserved intact, and the daughter molecule consists entirely of new chains.
• In the dispersive model, daughter molecules contain alternating segments of parental and newly synthesized sequences.

All three variants have been described in viruses, whereas only the semi-conservative mechanism operates in prokaryotes and eukaryotes. This was proven by Matthew Meselson and Franklin Stahl: a Escherichia coli culture was grown in a medium containing the heavy nitrogen isotope ¹⁵N, then transferred to a medium with ¹⁴N, and DNA density was analyzed by cesium chloride (CsCl) density gradient ultracentrifugation. After the first round, an intermediate-density band was observed; after the second, both intermediate and light bands appeared, confirming the semi-conservative mechanism.

Prokaryotic DNA polymerases

There are three main types of DNA polymerases in prokaryotes:

• DNA polymerase I removes RNA primers via 5′→3′ exonuclease activity and fills the resulting gaps.
• DNA polymerase II is involved in DNA damage repair.
• DNA polymerase III is the primary replication enzyme, highly processive, and responsible for synthesizing the new chain.

DNA polymerase III of E. coli has a complex multi-subunit structure:

• α subunit: catalytic, responsible for 5′→3′ DNA synthesis.
• ε subunit: 3′→5′ exonuclease activity for proofreading.
• θ subunit: complex stabilization, supports ε function.
• β subunit: forms a ring structure (sliding clamp) around DNA, ensuring high processivity.
• γ complex: clamp loader complex, hydrolyzes ATP, opens and closes the β ring.
• τ subunit: binds two catalytic cores, coordinates leading and lagging strand synthesis, and interacts with helicase.

Eukaryotic DNA polymerases

Five main polymerases are described in eukaryotes:

• Polymerase α: operates in a complex with primase; initiates synthesis by extending a short DNA stretch on the RNA primer.
• Polymerase δ: the main enzyme for lagging strand synthesis; functions in a complex with PCNA (proliferating cell nuclear antigen), which acts as a sliding clamp.
• Polymerase ε: synthesis of the leading strand and participation in repair.
• Polymerase β: base excision repair.
• Polymerase γ: mitochondrial DNA replication.

Telomeres and telomerase

Telomeres are the terminal regions of linear chromosomes, consisting of short tandem repeats. In humans, the repeat sequence is TTAGGG (three guanines, two thymines, and an adenine). Telomeres serve as a platform for placing the RNA primer and protect the ends from enzymatic degradation.

The end-replication problem arises because lagging strand synthesis requires an RNA primer, and there is not enough space to place one at the very end of a linear molecule. As a result, each division is accompanied by the loss of a short DNA fragment at the chromosome ends.

The solution is the enzyme telomerase, which possesses reverse transcriptase activity. It contains a protein catalytic component and an integrated RNA template complementary to the telomere repeat. Telomerase extends the repeats at the 3′ end; then endonucleases remove excess DNA, forming a characteristic T-loop where the 3′ end folds back into the double-stranded structure, stabilizing the chromosome.

Stages of replication

Replication is divided into three stages: initiation, elongation, and termination.

Initiation

• Recognition of the origin of replication: in prokaryotes, a single site is bound by DnaA proteins; in eukaryotes, multiple origins are recognized by the ORC complex with the participation of regulatory proteins CDC6 and CDT1, which license replication.
• Unwinding of the double helix by helicase to form the replication fork; single-stranded regions are stabilized by SSB proteins.
• Synthesis of RNA primers by primase; in eukaryotes, primase operates in a complex with polymerase α.

Elongation

• The leading strand is synthesized continuously in the 5′→3′ direction (Pol III in prokaryotes, Pol ε in eukaryotes).
• The lagging strand is synthesized discontinuously in short Okazaki fragments (Pol III in prokaryotes, Pol δ in eukaryotes), each starting with its own RNA primer.
• Removal of primers and joining of fragments: in prokaryotes, Pol I fills the gaps; in eukaryotes, primers are removed and gaps are filled by polymerase δ. DNA ligase covalently joins the fragments into a continuous chain.

Termination

• In prokaryotes, replication forks halt at terminal Ter sequences bound by the Tus protein.
• In eukaryotes, the forks merge with one another.
• Separation of daughter molecules is ensured by topoisomerases, which relieve supercoiling and untangle linked ring molecules or sister chromatids.

Okazaki experiments

In the 1960s–1970s, Reiji and Tsuneko Okazaki investigated the mechanism of DNA synthesis. Radiolabeled precursors (e.g., tritium-labeled thymidine) were added to the culture medium of E. coli infected with T4 bacteriophage. At short time intervals, DNA was isolated and separated by electrophoresis, and the distribution of labels was assessed by autoradiography.

Pulse samples revealed short-lived fragments about 1000 base pairs long, subsequently named Okazaki fragments. They form on the lagging strand, start with an RNA primer, and are then ligated into a continuous chain. This proved the discontinuous nature of the synthesis of one strand alongside the overall continuous nature of the other.

Key points

• DNA replication in prokaryotes and eukaryotes proceeds via a semi-conservative model, proven by the Meselson-Stahl experiment.
• In prokaryotes, the key enzyme is DNA polymerase III, while Pol I removes primers; in eukaryotes, the leading strand is synthesized by Pol ε, and the lagging strand by Pol δ.
• High processivity is provided by sliding clamps: the β ring in prokaryotes and PCNA in eukaryotes.
• Telomeres protect chromosome ends, and telomerase with its own RNA template solves the end-replication problem.
• Stages of replication: initiation (origin, helicase, SSB, primase), elongation (leading and lagging strands), termination (Ter/Tus, topoisomerases).
• The lagging strand is synthesized as Okazaki fragments, which are joined by DNA ligase.
• The proofreading 3′→5′ exonuclease activity of polymerases ensures high copying fidelity.
• Okazaki's experiments with radioactive labeling and electrophoresis proved the discontinuous synthesis of one DNA strand.