4.1 DNA replication

DNA replication is the process by which a DNA molecule makes an exact copy of itself before a cell divides. It takes place in the S phase of interphase, so that each daughter cell receives a complete set of genetic instructions. Replication is semi-conservative: each new double helix contains one original (parental) strand and one newly made strand. It is very accurate, with about one error in 10⁹ to 10¹⁰ bases after proofreading and repair, because each strand acts as a template and bases pair only A with T and G with C. Example: a plant disease diagnostic laboratory in Ol Joro Orok uses the polymerase chain reaction, a test-tube copy of DNA replication, to detect bacterial wilt in seed potato tubers.

Evidence for semi-conservative replication

Matthew Meselson and Franklin Stahl (1958) grew the bacterium Escherichia coli for many generations in a medium containing heavy nitrogen (¹⁵N), then moved it to normal nitrogen (¹⁴N). DNA from each generation was spun in a caesium chloride density gradient.

• Generation 0: All DNA formed one heavy band (¹⁵N/¹⁵N).
• Generation 1: All DNA formed one intermediate band (¹⁵N/¹⁴N), ruling out conservative replication.
• Generation 2: Half the DNA was intermediate and half light (¹⁴N/¹⁴N), ruling out dispersive replication.
• Conclusion: Each new molecule keeps one old strand and gains one new strand.

Enzymes and proteins of replication

Stages of DNA replication

1. Initiation: Replication starts at specific sites called origins of replication. Bacteria have one origin on their circular chromosome; a human chromosome has thousands, so the whole genome can be copied in a few hours.
2. Unwinding: Helicase separates the two strands, forming a Y-shaped replication fork. Single-strand binding proteins keep the strands apart and topoisomerase prevents supercoiling ahead of the fork.
3. Priming: Primase lays down a short RNA primer, about 10 nucleotides long, complementary to the template.
4. Elongation: DNA polymerase reads the template 3′ to 5′ and adds free deoxyribonucleotides (dATP, dGTP, dCTP, dTTP) to the 3′ end of the new strand, so new DNA always grows in the 5′ to 3′ direction. The energy comes from removing two phosphates from each incoming nucleotide.
5. Leading strand: On one template strand, the new strand is made continuously towards the replication fork.
6. Lagging strand: On the other template, the new strand is made discontinuously away from the fork in short pieces called Okazaki fragments (about 1,000 to 2,000 nucleotides in bacteria and 100 to 200 in eukaryotes), each starting from its own primer.
7. Primer removal and joining: DNA polymerase I removes the RNA primers and replaces them with DNA; DNA ligase seals the gaps between fragments.
8. Proofreading and termination: DNA polymerase checks each new base and removes mismatches (3′ to 5′ exonuclease activity). Replication ends when forks meet, and each daughter molecule rewinds into a double helix.

Differences between prokaryotic and eukaryotic replication

• Origins: One origin in bacteria; many origins in each eukaryotic chromosome.
• Speed: Bacterial forks move about 1,000 nucleotides per second; eukaryotic forks about 50 to 100 per second.
• Chromosome shape: Circular in bacteria, so there is no end problem; linear in eukaryotes, so telomeres shorten unless telomerase restores them.
• Histones: Eukaryotic DNA must also be repackaged into nucleosomes after replication.
• Polymerases: Eukaryotes use different polymerases (such as alpha, delta and epsilon) for priming, the lagging strand and the leading strand.

Polymerase chain reaction (PCR)

PCR copies a chosen section of DNA millions of times in a few hours, using the same principles as replication but driven by temperature changes.

1. Denaturation: Heat to about 95 °C to separate the DNA strands.
2. Annealing: Cool to about 50 to 65 °C so short DNA primers bind to each side of the target sequence.
3. Extension: Warm to 72 °C so a heat-stable DNA polymerase (Taq polymerase, from the hot-spring bacterium Thermus aquaticus) builds new strands from the primers.
4. Repeat: Each cycle doubles the target DNA. After n cycles, one molecule gives 2ⁿ copies.

Example calculation: number of copies from one DNA molecule after 30 cycles.

Copies = 2ⁿ

Copies = 2³⁰

Copies = 1,073,741,824, about one billion copies

Importance of accurate replication

• Genetic continuity: Every body cell carries the same genetic information as the zygote.
• Inheritance: Gametes pass accurate copies of genes to offspring.
• Mutation: Rare uncorrected errors are a source of new alleles and of disease.
• Drug targets: Some antiviral and anticancer drugs, such as zidovudine and certain chemotherapy agents, act by blocking DNA synthesis.