DNA Replication
The Importance of DNA Replication
Before a cell divides (by mitosis or meiosis), it must copy its entire genome so that each daughter cell receives a complete set of genetic information. DNA replication occurs during the S phase of the cell cycle and produces two identical copies of each DNA molecule. The process is described as semi-conservative because each new DNA molecule consists of one original (parental) strand and one newly synthesised strand.
The Meselson-Stahl Experiment
The semi-conservative model was confirmed by Meselson and Stahl (1958) in a classic experiment using E. coli:
1. Bacteria were grown for many generations in a medium containing heavy nitrogen (¹⁵N) — all DNA became labelled with ¹⁵N (heavy DNA)
2. Bacteria were transferred to a medium containing normal ¹⁴N (light nitrogen)
3. After one generation, DNA was extracted and centrifuged in a caesium chloride (CsCl) density gradient
4. All DNA appeared at an intermediate density — consistent with each molecule containing one heavy and one light strand (semi-conservative prediction)
5. After two generations, DNA appeared as two bands: one at intermediate density and one at light density — exactly as predicted by the semi-conservative model
This ruled out both the conservative model (which would show one heavy and one light band after one generation) and the dispersive model (which would show a single band at a different intermediate density after two generations).
The Mechanism of DNA Replication
Step-by-Step Process
1. Unwinding and Separation
The enzyme DNA helicase binds to the DNA at a specific origin of replication and moves along the molecule, breaking the hydrogen bonds between complementary base pairs. This separates the two antiparallel strands, creating a replication fork — a Y-shaped region where the two strands are being unwound.
- Single-strand binding proteins (SSBs) bind to the exposed single strands to prevent them from re-annealing (rejoining) or being degraded by nucleases
- The unwinding creates positive supercoiling ahead of the fork; topoisomerase (DNA gyrase in prokaryotes) relieves this tension by cutting and re-joining the DNA
2. Priming
DNA primase (an RNA polymerase) synthesises a short RNA primer (about 10-12 nucleotides) complementary to each template strand. This primer is necessary because DNA polymerase cannot initiate synthesis de novo — it can only add nucleotides to the 3' end of an existing strand.
3. Elongation by DNA Polymerase
DNA polymerase III (in prokaryotes; DNA polymerase δ and ε in eukaryotes) adds free DNA nucleotides to the 3' end of the primer/growing strand, using each original strand as a template. Nucleotides are added according to complementary base pairing rules:
- Adenine (A) pairs with Thymine (T) — 2 hydrogen bonds
- Guanine (G) pairs with Cytosine (C) — 3 hydrogen bonds
DNA polymerase always synthesises in the 5' to 3' direction (adding nucleotides to the 3' -OH end of the growing strand). Because the two template strands are antiparallel, replication proceeds differently on each strand:
- Leading strand — synthesised continuously in the 5'→3' direction, following the replication fork. Only one RNA primer is needed.
- Lagging strand — synthesised discontinuously in short segments called Okazaki fragments (1000-2000 nucleotides in prokaryotes, 100-200 in eukaryotes), each requiring its own RNA primer. The lagging strand runs in the opposite direction to fork movement.
4. Primer Removal and Gap Filling
DNA polymerase I (in prokaryotes; DNA polymerase δ in eukaryotes) removes the RNA primers and replaces them with DNA nucleotides, using the adjacent Okazaki fragment or leading strand as a primer.
5. Ligation
DNA ligase joins the Okazaki fragments on the lagging strand by catalysing the formation of phosphodiester bonds between the 3' end of one fragment and the 5' end of the next, creating a continuous strand.
Summary of Key Enzymes
| Enzyme | Function |
|---|---|
| DNA helicase | Unwinds double helix; breaks hydrogen bonds between base pairs |
| Single-strand binding proteins | Stabilise single-stranded DNA; prevent re-annealing |
| Topoisomerase | Relieves tension from supercoiling ahead of the fork |
| DNA primase | Synthesises short RNA primers |
| DNA polymerase III / δ, ε | Adds nucleotides 5'→3' using template; main replication enzyme |
| DNA polymerase I / δ | Removes RNA primers; fills gaps with DNA |
| DNA ligase | Joins Okazaki fragments; seals nicks in the sugar-phosphate backbone |
Accuracy and Proofreading
DNA replication is remarkably accurate, with an error rate of approximately 1 in 10⁹ nucleotides. This accuracy is achieved by:
- Complementary base pairing — the template dictates which nucleotide is added
- Proofreading by DNA polymerase — the enzyme has 3'→5' exonuclease activity, allowing it to detect a mismatched base, remove it, and replace it with the correct nucleotide
- Mismatch repair systems that scan newly replicated DNA for errors after replication
Eukaryotic Replication: Multiple Origins
Eukaryotic chromosomes are much longer than prokaryotic ones. To replicate the entire genome within S phase, replication begins simultaneously at multiple origins of replication along each chromosome. Replication proceeds bidirectionally from each origin, forming replication bubbles that expand and eventually merge, completing replication of the whole chromosome.
The End Replication Problem and Telomeres
At the ends of linear chromosomes, the removal of the final RNA primer on the lagging strand leaves a gap that cannot be filled (there is no upstream 3' -OH for DNA polymerase to extend from). This means that with each round of replication, chromosomes get slightly shorter at their ends.
Telomeres — repetitive, non-coding DNA sequences (TTAGGG in humans, repeated thousands of times) at chromosome ends — act as protective caps. They are progressively shortened with each division, acting as a mitotic clock. When telomeres become critically short, the cell enters senescence (G0) or undergoes apoptosis.
Telomerase is a reverse transcriptase enzyme that can extend telomeres by adding repetitive sequences. It is active in stem cells and germ cells (maintaining telomere length for indefinite division) but inactive in most somatic cells. Reactivation of telomerase in somatic cells is a hallmark of cancer, contributing to cellular immortality.
Exam Tips
- AQA frequently asks you to describe the roles of specific enzymes — always name the enzyme AND state its precise function
- Distinguish clearly between the leading strand (continuous) and lagging strand (discontinuous, Okazaki fragments)
- The Meselson-Stahl experiment is a classic AQA question — be able to describe the method and explain how results at generations 1 and 2 support the semi-conservative model
- Remember that DNA polymerase always works 5'→3' — this is why the lagging strand must be synthesised in fragments