# DNA replication

> CIE A-Level Biology · 9700 (2022 - )
> Source: https://www.owlsprep.com/study/cie-9700-u6-dna-replication/

This module covers the mechanism of DNA replication, the semi-conservative model, key enzyme roles, and experimental evidence, aligned to CIE A-Level Biology 9700 exam requirements.

**Prerequisites:** [DNA structure](https://www.owlsprep.com/study/cie-9700-u6-dna-structure/); [Nucleic acid structure](https://www.owlsprep.com/study/cie-9700-u6-nucleic-acid-structure/)

## Learning objectives

- Describe the semi-conservative model of DNA replication
- Outline the steps of DNA replication and roles of key enzymes
- Distinguish between leading and lagging strand synthesis
- Explain Meselson-Stahl experiment evidence for semi-conservative replication

## The Semi-Conservative Model of Replication

DNA replication is the process by which a DNA molecule makes an identical copy of itself, occurring before all forms of cell division (mitosis, meiosis, binary fission). The widely accepted semi-conservative model states that each new DNA molecule consists of one intact original parental strand and one newly synthesised complementary daughter strand.

**Semi-conservative replication** — A model of DNA replication where each resulting double helix retains one original parental strand and one newly built complementary strand.

*Example:* After one round of replication, every DNA molecule is a hybrid, containing one old and one new strand.

**Worked example:** Explain why conservative replication is ruled out after one round of replication in the Meselson-Stahl experiment.

1. Meselson and Stahl grew *E. coli* in heavy nitrogen ($^{15}$N) for many generations, so all parental DNA contained only heavy nitrogen.
2. They then transferred the bacteria to growth medium containing only light nitrogen ($^{14}$N) for one full generation of replication.
3. If replication were conservative, the original parental DNA would remain entirely heavy, and all new DNA would be entirely light, producing two separate bands after centrifugation.
4. The observed result was one single band of intermediate density, matching the prediction of semi-conservative replication (all hybrid DNA), so conservative replication is ruled out.

> **Exam tip:** Always explicitly state that each new DNA molecule retains one parental strand when describing semi-conservative replication — this is a common, easy to miss marking point.

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## Key Enzymes and Core Steps of Replication

Replication initiates at specific sequences called origins of replication, where the double helix is opened up. Multiple key enzymes coordinate the process, as outlined below:

- **DNA helicase**: Unwinds the double helix and breaks hydrogen bonds between complementary base pairs to separate the two strands, forming a replication fork.
- **Single-strand binding proteins**: Bind to the separated single strands to prevent them from reannealing (rewinding back into a double helix).
- **DNA primase**: Synthesises a short RNA primer that provides a free 3' end for DNA polymerase to start elongation.
- **DNA polymerase**: Adds complementary nucleotides to the growing new strand, moving along the template strand in the 3' → 5' direction.
- **DNA ligase**: Joins adjacent DNA fragments and seals nicks in the sugar-phosphate backbone after RNA primers are replaced with DNA.

**Replication fork** — The Y-shaped region where DNA is unwound and active replication of new strands is occurring.

**Worked example:** Why are RNA primers required for DNA replication?

1. DNA polymerase has a key structural limitation: it can only add nucleotides to an existing free 3'-OH end of a nucleic acid strand. It cannot start a new strand de novo (from scratch).
2. RNA primase solves this problem by synthesising a short (10-12 nucleotide) RNA primer that provides the required free 3' end for DNA polymerase to bind and start elongation.
3. After elongation is complete, the RNA primer is degraded and replaced with DNA by DNA polymerase, and any remaining nick in the backbone is sealed by DNA ligase.

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## Leading and Lagging Strand Synthesis

The two strands of the DNA double helix are antiparallel, so their 3' and 5' ends are oriented in opposite directions. Since DNA polymerase can only add nucleotides to the 3' end of a growing strand (synthesising new DNA 5' → 3'), replication proceeds differently on the two template strands.

**Okazaki fragments** — Short, discontinuous fragments of DNA synthesised on the lagging strand during replication.

The leading strand is synthesised continuously, because its template strand is oriented 3' → 5' towards the replication fork, so DNA polymerase can follow the moving fork directly. The lagging strand is synthesised discontinuously, because its template is oriented 5' → 3' towards the fork, so DNA polymerase has to work away from the fork in short sections, producing Okazaki fragments.

**Worked example:** Explain why the lagging strand is synthesised discontinuously.

1. Recall the core rule: DNA polymerase always reads the template strand 3' → 5' and builds the new strand 5' → 3', only adding to a free 3' end.
2. The two template strands at the replication fork are antiparallel, so one template has its 3' end at the fork, and the other has its 5' end at the fork.
3. For the template with 5' at the fork, DNA polymerase cannot move towards the fork (that would require synthesising new DNA 3' → 5', which it cannot do).
4. Instead, every time the replication fork opens up further, a new RNA primer is added, and DNA polymerase synthesises a short Okazaki fragment moving away from the fork. The fragments are later joined by ligase, resulting in a complete lagging strand. This discontinuous process is required because of DNA polymerase's directionality.

> **Exam tip:** Always remember the direction rule: DNA synthesised 5' → 3' always. Drawing a simple replication fork with labelled 3' and 5' ends in your answer can help you avoid mixing up leading and lagging strands in exam questions.

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## Meselson-Stahl Experimental Evidence

The Meselson-Stahl experiment (1958) provided definitive experimental evidence that DNA replication is semi-conservative, ruling out the alternative conservative and dispersive models of replication.

1. Bacteria were grown for many generations in medium containing only heavy $^{15}$N, so all bacterial DNA was uniformly heavy.
2. Bacteria were then transferred to medium containing only light $^{14}$N, and allowed to replicate for 1, 2, or more generations.
3. DNA was extracted from each generation, and its density was measured via density gradient centrifugation.
4. Results: 1 generation = 1 intermediate band (rules out conservative), 2 generations = 1 intermediate + 1 light band (rules out dispersive, confirms semi-conservative).

**Worked example:** Predict the DNA banding pattern after 3 generations of replication in the Meselson-Stahl experiment, starting from fully heavy DNA.

1. Generation 0: All DNA is heavy, one heavy band.
2. Generation 1: All DNA is hybrid (one heavy + one light strand), one intermediate band.
3. Generation 2: Half of DNA is hybrid, half is all light, so two bands: intermediate and light.
4. Generation 3: After 3 rounds of replication, there are 8 total DNA strands. Only 1 original heavy strand remains, paired with a light strand (hybrid), all other 7 strands are all light. This produces a faint intermediate band and a thick bright light band.

*Calculator:* forbidden

## Common pitfalls

- **Wrong:** Claiming helicase breaks phosphodiester bonds to unwind DNA
  - Why it fails: Confusion between bonds in the DNA backbone versus bonds between bases
  - Correct: Helicase breaks hydrogen bonds between complementary nitrogenous bases to separate the two strands
- **Wrong:** Saying DNA polymerase adds nucleotides to the 5' end of the growing strand
  - Why it fails: Mixing up template reading direction and new strand synthesis direction
  - Correct: DNA polymerase adds nucleotides to the free 3' end of the growing new strand, synthesising new DNA 5' → 3'
- **Wrong:** Claiming both semi-conservative and dispersive replication are ruled out after one generation
  - Why it fails: Confusing results of the first and second generation of the Meselson-Stahl experiment
  - Correct: Only conservative replication is ruled out after one generation; dispersive replication is ruled out after the second generation
- **Wrong:** Forgetting that DNA ligase is required on the lagging strand
  - Why it fails: Assuming Okazaki fragments automatically form a complete strand
  - Correct: Okazaki fragments are separate, and DNA ligase is required to join them into a single continuous strand
- **Wrong:** Describing DNA replication as conservative
  - Why it fails: Mixing up the names of the three replication models
  - Correct: DNA replication is semi-conservative: each new DNA retains one original parental strand

## Cheatsheet

| Component/Process | Key Fact for Exam |
| --- | --- |
| Semi-conservative replication | 1 parental strand + 1 new strand per new DNA |
| DNA helicase | Unwinds DNA, breaks H-bonds between bases |
| DNA primase | Makes RNA primer to start replication |
| DNA polymerase | Synthesises new DNA 5' → 3' |
| DNA ligase | Joins Okazaki fragments on lagging strand |
| Leading strand | Continuous synthesis, same direction as fork |
| Lagging strand | Discontinuous, made of Okazaki fragments |
| Meselson-Stahl Gen 1 | 1 intermediate band (rules out conservative) |
| Meselson-Stahl Gen 2 | 2 bands (intermediate + light, rules out dispersive) |

## What's next

DNA replication is the foundational process for cell division and genetic inheritance, and it is the first step in the central dogma of molecular biology. Mastering replication sets up understanding of transcription and translation, the processes that convert genetic information into functional proteins. This sub-topic is frequently tested alongside DNA structure and protein synthesis in both multiple choice and extended response questions for CIE 9700, and it also provides context for understanding mutations and genetic variation, key topics in further genetics study.

- [Transcription](https://www.owlsprep.com/study/cie-9700-u6-transcription/)
- [Translation](https://www.owlsprep.com/study/cie-9700-u6-translation/)
- [The genetic code](https://www.owlsprep.com/study/cie-9700-u6-the-genetic-code/)

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