Page 38 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

6.4 DNA Replication 197

Figure 6.20 How the Meselson-Stahl experiment confirmed semiconservative replication. (1) E. coli cells were grown in
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14
heavy N medium. (2) and (3) The cells were transferred to N medium and allowed to divide either once or twice. When DNA from each
of these cell preparations was centrifuged in a cesium chloride gradient, the density of the extracted DNA conformed to the predictions of
the semiconservative mode of replication, as shown at the bottom of the figure, where blue indicates heavy DNA and magenta depicts light
DNA. The results are inconsistent with the conservative and dispersive models for DNA replication (compare with Fig. 6.19).

14 N 15 N 14 N 14 N

Control: E. coli grown for many 1. E. coli grown for many 2. Cells replicate once to 3. Cells replicate a second
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generations in N medium. generations in N medium. 30 produce first generation 30 time to produce a second
minutes minutes
of daughter cells. generation of daughter cells.
Extract DNA from cells. Extract DNA from cells. Extract DNA from cells. Extract DNA from cells.

Centrifuge Centrifuge Centrifuge Centrifuge

DNA bands in cesium chloride gradient

14 N N
14
14 N N
14
14
15 14 15 N N
N N
15 N N
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Results support hypothesis of semiconservative replication.
mitosis, each of the two daughter cells receives one sister replication machinery from E. coli bacteria. Remarkably,
chromatid from every chromosome in the cell. This process they were eventually able to elicit the reproduction of spe-
preserves chromosome number and identity during mitotic cific genetic information outside a living cell, in a test tube
cell division because the two sister chromatids are identical containing purified enzymes together with a DNA template,
in base sequence to each other and to the original parental primers (defined below), and nucleotide triphosphates.
chromosome. Although the biochemistry of DNA replication was elu-
cidated for a single bacterial species, its essential features are
DNA Polymerase Has Strict Operating conserved—just like the structure of DNA—within all
Requirements organisms. The energy required to synthesize every DNA
molecule found in nature comes from the high-energy phos-
Watson and Crick’s model for semiconservative replication phate bonds associated with the four deoxyribonucleotide
is a simple concept to grasp, but the biochemical process triphosphates (dATP, dCTP, dGTP, and dTTP; or dNTP as a
through which it occurs is quite complex. Replication does general term) that provide bases for incorporation into the
not happen spontaneously any time a mixture of DNA and growing DNA strand. As shown in Fig. 6.21, this conserved
nucleotides is present. Rather, it occurs at a precise moment biochemical feature means that DNA synthesis can proceed
in the cell cycle, depends on a network of interacting regula- only by adding nucleotides to the 3′ end of an existing poly-
tory elements, requires considerable input of energy, and in- nucleotide. With energy released from severing the triphos-
volves a complex array of the cell’s molecular machinery, phate arm of a dNTP substrate molecule, the DNA
including the key enzyme DNA polymerase. The sa- polymerase enzyme catalyzes the formation of a new pho-
lient details were deduced primarily in the laboratory of sophodiester bond. Once this bond is formed, the enzyme
Arthur Kornberg, who won a Nobel prize for this work. proceeds to join up the next nucleotide brought into position
The Kornberg group purified individual components of the by complementary base pairing.