DNA replication

The essentials

While proposing the double helix, Watson and Crick also suggested that the two strands would separate and each would act as a template for a new complementary strand. Each daughter DNA would therefore have one parental and one new strand. This is semi-conservative replication.

Matthew Meselson and Franklin Stahl (1958) proved it with E. coli. They grew bacteria for many generations in a medium with ¹⁵NH₄Cl, so the DNA became "heavy" (¹⁵N). They then shifted the cells to a medium with normal ¹⁴NH₄Cl and took samples as the cells divided (about every 20 minutes). DNA was separated by caesium chloride (CsCl) density gradient centrifugation. After one generation (20 minutes), all DNA had an intermediate (hybrid) density. After two generations (40 minutes), there were equal amounts of hybrid and light DNA. Similar results were later obtained by Taylor and colleagues using radioactive thymidine on Vicia faba (faba bean) chromosomes.

Machinery: the main enzyme is DNA-dependent DNA polymerase, which uses a DNA template to add deoxyribonucleotides. In E. coli, which has about 4.6 × 10⁶ bp, replication finishes within about 18 minutes, so the polymerase is very fast and accurate. Deoxyribonucleoside triphosphates act both as substrates and as the energy source. Replication begins at a specific point called the origin of replication. The two strands are opened at a small region forming the replication fork. DNA polymerase works only in the 5′→3′ direction. So, on one template (3′→5′) synthesis is continuous (leading strand), and on the other template (5′→3′) it is discontinuous (lagging strand), producing short fragments that are joined by DNA ligase.

Key points

• In eukaryotes, replication takes place in the S phase of the cell cycle.
• A vector needs an origin of replication to propagate foreign DNA.

Common mistakes

• DNA polymerase cannot start a new strand in the 3′→5′ direction; that is why the lagging strand forms in fragments.