DNA replication is the process by which a cell duplicates its genome prior to cell division. In eukaryotes, replication occurs during the S phase of the cell cycle and is initiated at thousands of origin sequences simultaneously.
Note: The accuracy of eukaryotic DNA replication is remarkable — after proofreading and mismatch repair, the error rate is approximately 1 error per 10 billion base pairs.
Helicase (MCM2–7 complex in eukaryotes) unwinds parental duplex DNA by breaking hydrogen bonds:
- Proceeds bidirectionally from each origin of replication
- Requires ATP hydrolysis (~2 ATP per base pair unwound)
- Creates a replication fork structure with two template strands
Eukaryotes employ three primary replicative polymerases:
| Polymerase | Role | Processivity |
|---|---|---|
| Pol α / Primase | Synthesizes RNA primers | Low |
| Pol δ | Lagging strand synthesis | High (with PCNA) |
| Pol ε | Leading strand synthesis | Very high |
Key properties of DNA Polymerases:
- Synthesizes DNA exclusively in 5' to 3' direction
- Requires a free 3'-OH group (primer) to initiate
- 3' to 5' exonuclease activity provides proofreading
Warning: Defects in DNA Pol epsilon's proofreading domain (POLE mutations) are a driver of hypermutated colorectal and endometrial cancers — these tumors have over 100 times the normal mutation burden.
Because DNA polymerase can only extend in the 5' to 3' direction, synthesis is discontinuous on the lagging strand:
- Primase lays down a short RNA primer (~10 nt)
- Pol δ extends the primer creating an Okazaki fragment (~150–200 nt in eukaryotes)
- RNase H / FEN1 removes RNA primers
- Pol δ fills the gap with DNA
- DNA Ligase I seals the nick with a phosphodiester bond
A fundamental limitation: conventional DNA polymerase cannot fully replicate chromosome ends (the "end-replication problem"). Each round of replication shortens telomeres by ~50–200 bp.
Solution — Telomerase:
- A specialized reverse transcriptase with an internal RNA template (TERC)
- Extends the 3' telomeric overhang using its RNA template
- Expressed in germline cells, stem cells, and most cancer cells
Tip: Telomerase is upregulated in ~85% of human cancers, making it an attractive therapeutic target. BIBR1532 and other small-molecule inhibitors are in clinical trials.