When Watson and Crick published their paper describing the structure of DNA, they also coyly laid claim to another discovery: “It has not escaped our notice that the specific pairing we have postulated [A with T, and G with C] immediately suggests a copying mechanism for the genetic material.” The copying mechanism they had in mind is exquisitely simple. The two strands of the parental duplex molecule separate (Fig. 12.1), and each individual parental strand serves as a model, or template strand, for the synthesis of a daughter strand. As each daughter strand is synthesized, the order of the bases in the template strand determines the order of the complementary bases added to the daughter strand. For example, the sequence 5′-ATGC-3′ in the template strand specifies the sequence 3′-TACG-5′ in the daughter strand because A pairs with T and G pairs with C. (The designations 3′ and 5′ convey the antiparallel orientation of the strands.)

A key prediction of the model shown in Fig. 12.1 is semiconservative replication. That is, after replication, each new DNA duplex consists of one strand that was originally part of the parental duplex and one newly synthesized strand. An alternative model is conservative replication, which proposes that the original DNA duplex remains intact and the daughter DNA duplex is completely new. Which model is correct? If there were a way to distinguish newly synthesized daughter DNA strands (“new strands”) from previously synthesized parental strands (“old strands”), the products of replication could be observed and the mode of replication determined.

American molecular biologists Matthew S. Meselson and Franklin W. Stahl carried out an experiment to determine how DNA replicates. This experiment, described in Fig. 12.2, has been called “the most beautiful experiment in biology” because it so elegantly demonstrated the scientific method (Chapter 1) of hypothesis, prediction, and experimental test. They distinguished “old” from “new” DNA strands by labeling them with nonradioactive isotopes of nitrogen that have different densities: the normal form of nitrogen, ¹⁴N, and a heavier form with an extra neutron, denoted ¹⁵N.

Meselson and Stahl found that DNA in fact replicates semiconservatively (Fig. 12.2). This finding also predicted the results when cells are allowed to undergo two rounds of replication in a medium containing only light ¹⁴N nitrogen. The heavy strand and light strand each serve as templates for a new light daughter strand. The result is that half of the DNA molecules will have one heavy old strand and one light new strand and an intermediate density, and half of the DNA molecules will have one light old strand and one light new strand and a low density. This is precisely what they observed (Fig. 12.2).

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Quick Check 1 Suppose Meselson and Stahl had done their experiment the other way around, starting with cells fully labeled with ¹⁴N light DNA and then transferring them to medium containing only ¹⁵N heavy DNA. What density of DNA molecule would you predict after one and two rounds of replication?

Important as it was in demonstrating semiconservative replication in bacteria, the Meselson–Stahl experiment left open the possibility that DNA replication in eukaryotes might be different. It was only some years after the Meselson–Stahl experiment that methods for labeling DNA with fluorescent nucleotides were developed. These methods allowed researchers to visualize entire strands of eukaryotic DNA and follow each strand through replication. Fig. 12.3 shows a human chromosome with unlabeled DNA that subsequently underwent two rounds of replication in medium containing a fluorescent nucleotide. The chromosome was photographed at metaphase of the second round of mitosis, after chromosome duplication but before the separation of the chromatids into the daughter cells. Notice that one chromatid contains hybrid DNA with one labeled strand and one unlabeled strand, which fluoresces faintly (light); the other chromatid contains two strands of labeled DNA, which fluoresces strongly (dark). This result is conceptually the same as what was seen by Meselson and Stahl after two rounds of replication and exactly as predicted by the semiconservative replication model. The result also demonstrates that each eukaryotic chromosome contains a single DNA molecule that runs continuously all along its length.