DNA replication is the process by which a cell copies its DNA before cell division. It ensures that each daughter cell receives an identical copy of the genetic information.
DNA replication is described as semi-conservative because each new DNA molecule consists of:
- One original (parental) strand
- One newly synthesized (daughter) strand
This model was proven by the Meselson-Stahl Experiment (1958).
- Bacteria were grown in a medium containing heavy nitrogen (15N) until all DNA was labelled with 15N.
- Bacteria were then transferred to a medium containing light nitrogen (14N).
- After one generation, DNA was extracted and centrifuged. Only hybrid (15N/14N) DNA was found — intermediate density.
- After two generations, both hybrid and light (14N/14N) DNA were found in a 1:1 ratio.
This result was only consistent with the semi-conservative model, ruling out conservative and dispersive models.
- Replication begins at specific sequences called origins of replication.
- Helicase unwinds the double helix by breaking the hydrogen bonds between complementary base pairs, creating a replication fork.
- Topoisomerase relieves the tension (supercoiling) ahead of the replication fork.
- Single-Strand Binding Proteins (SSBPs) stabilize the separated strands.
- DNA Polymerase cannot start a new strand from scratch; it can only extend an existing strand.
- Primase (an RNA polymerase) synthesizes a short RNA primer (~10 nucleotides) complementary to the template strand.
- The primer provides the free 3′-OH end that DNA Polymerase III requires to begin adding DNA nucleotides.
- DNA Polymerase III is the main replicative enzyme. It adds complementary deoxyribonucleotides to the 3′-OH end of the growing strand.
- Synthesis always proceeds in the 5′→3′ direction.
- Because the two template strands are antiparallel, the two new strands are synthesized differently:
| Strand | Template Direction | Synthesis | Mode |
|---|
| Leading strand | 3′→5′ toward fork | 5′→3′ | Continuous |
| Lagging strand | 5′→3′ away from fork | 5′→3′ | Discontinuous |
Because the lagging strand template runs away from the fork, DNA Polymerase III must repeatedly start new segments. These short, discontinuous segments are called Okazaki fragments.
- DNA Polymerase I removes the RNA primers using its 5′→3′ exonuclease activity.
- It simultaneously fills the gaps left by the removed primers with DNA nucleotides.
- DNA Ligase seals the nicks between adjacent Okazaki fragments (and between the filled gaps and existing DNA) by forming phosphodiester bonds.
- This produces a continuous, intact lagging strand.
| Enzyme | Function |
|---|
| Helicase | Unwinds double helix; breaks hydrogen bonds |
| Topoisomerase | Relieves supercoiling ahead of fork |
| Primase | Synthesizes RNA primers |
| DNA Polymerase III | Main enzyme; synthesizes new DNA (5′→3′) |
| DNA Polymerase I | Removes RNA primers; fills gaps with DNA |
| DNA Ligase | Joins Okazaki fragments; seals nicks |
- Replication Fork: The Y-shaped region where the double helix is unwound and new strands are synthesized.
- Origin of Replication: Specific DNA sequence where replication begins.
- Okazaki Fragments: Short DNA segments synthesized discontinuously on the lagging strand.
- RNA Primer: Short RNA sequence that provides the starting point for DNA synthesis.