DNA replication: the model and the machinery

Watson and Crick (1953) suggested how DNA copies itself: the two strands separate, and each acts as a template for a new partner, built by base pairing (A with T, G with C). Each new DNA molecule keeps one old strand and one new strand: semiconservative replication.

Meselson and Stahl (1958) proved it. They grew E. coli for many generations in heavy nitrogen (¹⁵N), so all their DNA was heavy, then moved them to normal light nitrogen (¹⁴N). They separated DNA by weight, spinning it in a caesium chloride density gradient.

• After one generation: a single band of intermediate density (one heavy strand + one light strand).
• After two generations: one intermediate band and one light band.

This is exactly what semiconservative replication predicts. Taylor, working with the bean plant Vicia faba, showed the same for whole chromosomes.

The enzymes that build DNA, DNA polymerases, can add nucleotides only in the 5' → 3' direction. In E. coli, polymerase III is the main copier, and it proof-reads its work (removing wrong nucleotides with its 3' → 5' exonuclease activity). Polymerase I removes RNA primers and fills the gaps. Polymerase II works in repair.

Other helpers at the replication fork:

• Helicase unzips the two strands by breaking the hydrogen bonds between them, using ATP.
• Primase makes a short RNA primer (about 10-12 nucleotides), because DNA polymerase cannot start a new chain on its own.
• Topoisomerase cuts and rejoins DNA to release the twisting that builds up ahead of the fork.
• Single-strand binding proteins keep the separated strands apart.
• DNA ligase seals gaps with phosphodiester bonds.