Formation of the Replication Fork
Preparation for synthesis begins by relieving torsional strain in the coiled DNA molecule. To achieve this, DNA topoisomerase (acting as a reversible nuclease) cleaves the 3',5'-phosphodiester bond of one strand and transiently attaches to the 5'-end, thereby relieving chromatin supercoiling.
Next, two molecules of DNA helicase utilize ATP hydrolysis energy to locally unwind the double helix. To prevent the unwound template strands from re-annealing (renaturation) or forming hairpin loops, SSB proteins (single-stranded DNA-binding proteins) attach to them. They keep the strands apart without covering the nitrogenous bases so that polymerases can 'read' them.
Synthesis of Leading and Lagging Strands
Daughter DNA strands are built antiparallel to the parental strands (templates are read 3' to 5'). Because of this, synthesis differs fundamentally between the two strands:
- Leading strand: Built continuously, following the moving replication fork. Synthesis here is driven by DNA polymerase $\delta$.
- Lagging strand: Forced to synthesize in the direction opposite to fork movement. Synthesis occurs discontinuously in short segments known as Okazaki fragments. Each fragment consists of a short RNA primer (about 10 nucleotides) and a DNA segment (about 150 nucleotides).
Polymerases require a starting point to initiate synthesis. This role is played by an RNA primer created by primase (a subunit of DNA polymerase $\alpha$). The enzyme then shifts to chain elongation before handing off the process to polymerase $\delta$ or $\epsilon$.
Fragment Ligation and Error Proofreading
On the lagging strand, RNA primers must be removed and the separate fragments joined into a single continuous strand. Primers are removed by endonucleases or RNases, and the resulting gaps are filled by DNA polymerase $\beta$. Finally, DNA ligase uses ATP to seal the fragments, restoring the phosphodiester backbone.
Quality control is a critical step. DNA polymerases $\delta$ and $\epsilon$ possess 3'-5' exonuclease activity. If they detect an incorrectly incorporated nucleotide, they excise it, ensuring high fidelity in duplicating genetic material.
Regulation and Eukaryotic Features
Because eukaryotic DNA molecules are massive, replication initiates not at a single site, but at multiple origins of replication. The region between adjacent origins is called a replicon. At each origin, two replication forks form and diverge in opposite directions until they collide with forks from neighboring replicons.
Initiation of replication is tightly controlled by cyclins. For instance, cyclin D drives cell entry into the S phase, while cyclins E and A trigger DNA synthesis itself. They activate cyclin-dependent kinases (CDKs), which phosphorylate the proteins required to progress through the cell cycle.