FIG. 12.2

How is DNA replicated?

BACKGROUND Watson and Crick’s discovery of the structure of DNA in 1953 suggested a mechanism by which DNA is replicated. Experimental evidence came from research by American molecular biologists Matthew Meselson and Franklin Stahl in 1958.

HYPOTHESIS DNA replicates in a semiconservative manner, meaning that each new DNA molecule consists of one parental strand and one newly synthesized strand.

ALTERNATIVE HYPOTHESIS DNA replicates in a conservative manner, meaning that one DNA molecule consists of two parental strands, and the other consists of two newly synthesized strands.

METHOD Meselson and Stahl distinguished parental strands (“old”) from newly synthesized strands (“new”) using two isotopes of nitrogen atoms. Old strands were labeled with a heavy form of nitrogen with an extra neutron (¹⁵N), and new strands were labeled with the normal, lighter form of nitrogen (¹⁴N).

EXPERIMENT The researchers first grew bacterial cells on medium containing only the heavy ¹⁵N form of nitrogen. As the cells grew, ¹⁵N was incorporated into the DNA bases, resulting, after several generations, in DNA containing only ¹⁵N. They then transferred the cells into medium containing only light ¹⁴N nitrogen. After one round of replication in this medium, cell replication was halted. The researchers could not observe the DNA directly, but instead they measured the density of the DNA by spinning it in a high-speed centrifuge in tubes containing a solution of cesium chloride.

PREDICTION If DNA replicates conservatively, half of the DNA in the cells should be composed of two heavy parental strands containing¹⁵N, and half should be composed of two light daughter strands containing ¹⁴N. If DNA replicates semiconservatively, the daughter DNA molecules should each consist of one heavy strand and one light strand.

RESULTS When the fully¹⁵N-labeled parental DNA was spun, it concentrated in a single heavy band with a density of 1.722 gm/cm³. After one round of replication, the DNA formed a band at a density of 1.715 gm/cm³, which is the density expected of a duplex molecule containing one heavy (¹⁵N-labeled) strand and one light (¹⁴N-labeled) strand. DNA composed only of ¹⁴N would have a density of 1.708 gm/cm³. After two rounds of replication, half the molecules exhibited a density of 1.715 gm/cm³, indicating a duplex molecule with one heavy strand and one light strand, and the other half exhibited a density of 1.708 gm/cm³, indicating a duplex molecule containing two light strands.

CONCLUSION DNA replicates semiconservatively, supporting the first hypothesis.

SOURCE Meselson, M., and F. W. Stahl. 1958. “The Replication of DNA in Escherichia coli.” PNAS 44:671–82.