Replication continues (b)
Figure 5.2. DNA polymerase III can correct its own mistakes.
polymerase III until it reaches the 5' end of the next RNA primer (Fig. 5.1). In prokaryotes the lagging strand is primed about every 1000 nucleotides whereas in eukaryotes this takes place each 200 nucleotides.
Once the synthesis of the DNA fragment is complete, the RNA primers must be replaced by deoxyribonucleotides. In prokaryotes the enzyme DNA polymerase I removes ribonucleotides using its 5' ^ 3' exonuclease activity and then uses its 5' ^ 3' polymerizing activity to incorporate deoxyribonucleotides. In this way, the entire RNA primer gets replaced by DNA. Synthesis of the lagging strand is completed by the enzyme DNA ligase, which joins the DNA fragments together by catalyzing formation of phosphodiester links between adjacent fragments.
Eukaryotic organisms probably use an enzyme called ribonuclease H to remove their RNA primers. This enzyme breaks phosphodiester links in an RNA strand that is hydrogen-bonded to a DNA strand.
The human genome consists of about 3 x 109 base pairs of DNA. DNA polymerase III makes a mistake about every 1 in 104 bases and joins an incorrect deoxyribonucleotide to the growing chain. If unchecked, these mistakes would lead to a catastrophic mutation rate. Fortunately, DNA polymerase III has an built-in proofreading mechanism that corrects its own errors. If an incorrect base is inserted into the newly synthesized daughter strand, the enzyme recognizes the change in shape of the double-stranded molecule, which arises through incorrect base pairing, and DNA synthesis stops (Fig. 5.2b). DNA polymerase III then uses its 3' to 5' exonuclease activity to remove the incorrect deoxyribonucleotide and replace it with the correct one. DNA synthesis then proceeds. DNA polymerase III hence functions as a self-correcting enzyme.
In 1958 Matthew Meselson and Franklin Stahl designed an ingenious experiment to test whether each strand of the double helix does indeed act as a template for the synthesis of a new strand. They grew the bacterium Escherichia coli in a medium containing the heavy isotope 15N that could be incorporated into new DNA molecules. After several cell divisions they transferred the bacteria, now containing "heavy" DNA, to a medium containing only the lighter, normal, isotope 14N. Any newly synthesized DNA molecules would therefore be lighter than the original parent DNA molecules containing 15N. The difference in density between the heavy and light DNAs allows their separation using very high speed centrifugation. The results of this experiment are illustrated in the figure. DNA isolated from cells grown in the 15N medium had the highest density and migrated the furthest during centrifugation. The lightest DNA was found in cells grown in the 14N medium for two generations, whereas DNA from bacteria grown for only one generation in the lighter 14N medium had a density half way between these two. This is exactly the pattern expected if each strand of the double helix acts as a template for the synthesis of a new strand. The two heavy parental strands separated during replication, with each acting as a template for a newly synthesized light strand, which remained bound to the heavy strand in a double helix. The resulting DNA was therefore of intermediate density. Only in the second round of DNA replication, when the light strands created during the first round of replication were allowed to act as templates for the construction of complementary light strands, did DNA double helices composed entirely of 14N-containing building blocks appear.
The two scientists were awarded the Nobel prize for this discovery that DNA replication is "semiconservative," meaning that the results are not completely new but are half new and half old.
bacteria grown in medium enriched with the heavy isotope 15N
bacteria transferred to medium containing 14N
bacteria continue to grow in medium containing 14N
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