Semi-conservative DNA replication

DNA replication copies a DNA molecule before a cell divides. The two strands of the original double helix separate, and each strand guides the formation of a new complementary strand. This produces two DNA molecules, each containing one original strand and one newly synthesised strand.

Separating the DNA strands

The DNA double helix unwinds, and the enzyme DNA helicase breaks the hydrogen bonds between complementary bases. The two polynucleotide strands separate, exposing their bases. Each original strand can now act as a template for a new strand.

Building the new strands

Free DNA nucleotides pair with the exposed bases on each template strand. The pairing is specific: adenine pairs with thymine, and cytosine pairs with guanine. The sequence of bases on each original strand therefore determines the sequence of bases in its new complementary strand.

The enzyme DNA polymerase catalyses condensation reactions that join adjacent nucleotides in each new strand. This forms the sugar-phosphate backbone of the new polynucleotide strand.

Why replication is semi-conservative

Each resulting DNA molecule contains one strand from the original molecule and one new strand. Replication is called semi-conservative because half of the original molecule—the original strand—is retained in each resulting molecule.

Complementary base pairing allows the base sequence to be copied. The resulting DNA molecules can therefore pass the same genetic information to the next generation of cells.

Experimental evidence for semi-conservative DNA replication

Watson and Crick proposed that the two strands of a DNA molecule separate during replication. Each original strand acts as a template for a new complementary strand. The result is two DNA molecules, each containing one original strand and one newly made strand. This is called semi-conservative replication.

Meselson and Stahl tested this prediction by labelling DNA according to its density. They grew bacteria in a medium containing heavy nitrogen, $$\ce{^{15}N}$$, so that the nitrogen incorporated into their DNA was heavy. They then transferred the bacteria to a medium containing light nitrogen, $$\ce{^{14}N}$$. Any new DNA strands made after the transfer contained light nitrogen.

The scientists extracted DNA after successive generations and used density-gradient centrifugation to separate it by density. DNA of different densities formed distinct bands: heavy DNA, light DNA and DNA of intermediate density could therefore be distinguished.

DNA after one generation

After one generation in light nitrogen, all the DNA formed one intermediate-density band. This was consistent with semi-conservative replication: each heavy original strand had served as a template for a new light strand, producing DNA molecules with one heavy and one light strand. These are called hybrid molecules.

The result did not support a conservative model. In that model, the original heavy molecule would remain intact and an entirely new light molecule would be made. It therefore predicted separate heavy and light bands, rather than a single intermediate band.

The first-generation result alone did not distinguish semi-conservative replication from a dispersive model. The dispersive model also predicted one intermediate band, but proposed that each strand contained a mixture of old and new DNA sections.

DNA after two generations

After a second generation in light nitrogen, the DNA formed two bands: one intermediate and one light. Semi-conservative replication predicted this result. Each hybrid molecule’s heavy strand produces another hybrid molecule, while its light strand produces a molecule containing two light strands.

The dispersive model instead predicted a single band that became progressively lighter as more light DNA was incorporated. The observed intermediate and light bands therefore supported semi-conservative replication over the dispersive model.

The evidence shows how old and new DNA are distributed between molecules after replication. The density patterns do not, by themselves, establish the mechanisms of the individual enzymes involved.