DNA replication

The elucidation of the structure of DNA by Watson and Crick in 1953 stands as one of the great scientific breakthroughs of the 20th century. An important implication of their model lay in the complementary nature of the two strands, as they commented themselves in their famous paper in Nature:

It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.

The mechanism they proposed became known as the semiconservative replication of DNA, so called because each daughter molecule comprises one parental strand and one newly synthesised strand (Figure 11.1). The parental double helix of DNA unwinds and each strand acts as a template for the production of a new complementary strand, with new nucleotides being added according to the rules of base-pairing. In 1957, 4 years after the publication of Watson and Crick's model, Matthew Meselson and Franklin Stahl provided experimental proof of the semiconservative nature of DNA replication (Box 11.2). The exact way in which replication takes place in procaryotic and eucaryotic cells differs in some respects, but we shall take as our model replication in E. coli, since it has been studied so extensively.

Box 11.2 Experimental proof of semiconservative replication

The semiconservative model of DNA replication, as suggested by Watson and Crick, was not the only one in circulation during the 1950s. Meselson and Stahl provided experimental evidence which not only supported the semiconservative model, but also showed the other models to be unworkable. Their elegantly designed experiment used newly developed techniques to differentiate between parental DNA and newly synthesised material.

First, they grew E. coli in a medium with ammonium salts containing the heavy isotope ,5N as the only source of nitrogen. This was done for several generations of growth, so that all the cells contained nitrogen exclusively in the heavy form.

The bacteria were then transferred to a medium containing nitrogen in the normal, ,4N form. After a single round of replication, DNA was isolated from the culture and subjected to density gradient centrifugation. This is able to differentiate between DNA containing the two forms of nitrogen, as ,5N has a greater buoyant density and therefore settles at a lower position in the tube.

Meselson and Stahl found just a single band of DNA after centrifugation, with a density intermediate between that of ,4N and ,5N, indicating a hybrid molecule, as predicted by the semiconservative model. After a second round of E. coli replication in a medium containing ,4N, two bands of DNA were produced, one hybrid and one containing exclusively ,4N, exactly as predicted by the semiconservative model, but inconsistent with other hypotheses.

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