Determining the DNA Replication Mechanism

The mechanism of DNA replication that had suggested itself to Watson and Crick was soon confirmed. First, experiments showed that single strands of DNA could be replicated in a test tube containing simple substrates and an enzyme. Then an elegant experiment showed that each of the two strands of the double helix serves as a template for a new strand of DNA.

Dispersive replication would produce two molecules with old and new DNA interspersed along each strand.

11.8 Three Models for DNA Replication In each model, original DNA is shown in blue and newly synthesized DNA in red.

new molecules, each containing old and new parts, perhaps at random (Figure 11.8c)

Watson and Crick's original paper suggested that DNA replication was semiconservative, but Kornberg's experiment did not provide a basis for choosing among these three models.

Meselson and Stahl demonstrated that DNA replication is semiconservative

A clever experiment conducted by Matthew Meselson and Franklin Stahl convinced the scientific community that semi-conservative replication is the correct model. Working at the California Institute of Technology in 1957, they devised a simple way to distinguish old strands of DNA from new ones: density labeling.

The key to their experiment was the use of a "heavy" isotope of nitrogen. Heavy nitrogen (15N) is a rare, nonradioac-tive isotope that makes molecules containing it more dense than chemically identical molecules containing the common isotope, 14N. To distinguish DNA of different densities (that is, DNA containing 15N versus DNA containing 14N), Meselson, Stahl, and Jerome Vinograd developed a new procedure using a centrifuge. Spinning solutions or suspensions at high speed in a centrifuge causes the solutes or particles to separate and form a gradient according to their density.

Meselson and Stahl grew two cultures of the bacterium Escherichia coli for many generations:

► One culture was grown in a medium whose nitrogen source (ammonium chloride, NH4Cl) was made with 15N instead of 14N. As a result, all the DNA in the bacteria was "heavy."

► Another culture was grown in a medium with 14N, and all the DNA in these bacteria was "light."

When extracts from the two cultures were combined and cen-trifuged, two separate DNA bands formed, showing that this method could distinguish DNA samples of slightly different densities.

Next, the researchers grew another E. coli culture on 15N medium, then transferred it to normal 14N medium and allowed the bacteria to continue growing (Figure 11.9). Under

11.9 The Meselson-Stahl Experiment A centrifuge was used to separate DNAs labeled with isotopes of different den-sities.This experiment revealed a pattern that supports the semiconservative model of DNA replication.

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