DNA Replication

Replication fork formation

Preparation for synthesis begins by relieving tension in the supercoiled DNA molecule. To do this, the enzyme DNA topoisomerase (acting as a reversible nuclease) cleaves the 3',5'-phosphodiester bond of one of the strands and temporarily attaches to the 5' end. This removes chromatin supercoiling.

Next, two molecules of DNA helicase come into play. Utilizing ATP energy, they locally unwind the double helix. To prevent the unwound template strands from re-annealing (renaturation) or forming hairpins, SSB proteins bind to them. They keep the strands apart without blocking the nitrogenous bases themselves, allowing polymerases to "read" them.

Leading and lagging strand synthesis

Daughter DNA strands are built antiparallel to the parental strands (templates are read from 3' to 5'). Because of this, the synthesis process on the two strands differs fundamentally.

• Leading strand: synthesized continuously following the moving replication fork. Synthesis here is carried out by DNA polymerase $\delta$.
• Lagging strand: forced to be synthesized in the direction opposite to the fork movement. Synthesis proceeds discontinuously, in pieces known as Okazaki fragments. Each such fragment consists of a short RNA primer (about 10 nucleotides) and a DNA segment (about 150 nucleotides).

To initiate synthesis, polymerases need a starting point. This role is played by an RNA primer created by primase (a subunit of DNA polymerase $\alpha$). The enzyme then switches to chain elongation and subsequently hands off the task to polymerase $\delta$ or $\epsilon$.

Fragment ligation and error correction

On the lagging strand, it is necessary to remove the RNA primers and join the separate pieces into a single strand. Primers are removed by endonucleases or RNases, and the resulting gaps are filled by DNA polymerase $\beta$. Finally, DNA ligase, using ATP, ligates the fragments, restoring the phosphodiester backbone.

Quality control is a crucial step. DNA polymerases $\delta$ and $\epsilon$ possess exonuclease activity. If they detect an incorrectly incorporated nucleotide, they excise it, ensuring high fidelity in the duplication of genetic material.

Regulation and eukaryotic features

In eukaryotes, DNA molecules are enormous, so the process begins not at a single point, but at multiple origins (initiation sites). The segment between adjacent origins is called a replicon. Two forks form at each origin and move in opposite directions until they collide with the forks of the neighboring replicon.

The initiation of replication is tightly controlled by cyclins. For example, cyclin D is responsible for the cell's transition into the S-phase, while cyclins E and A trigger DNA synthesis itself. They activate cyclin-dependent kinases, which phosphorylate proteins required for cell cycle progression.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Detailed mechanism of DNA topoisomerase action
• Cyclin specificity at various stages of the cell cycle
• Polymerase proofreading mechanisms
• The role of magnesium ions in the active site of polymerases
• Replicon structure and protein interactions within the complex

Other topics in the section "Template Biosyntheses"

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