Cell Biology. Stephen R. Bolsover

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Figure 4.2. DNA polymerase III can correct its own mistakes.

      Medical Relevance 4.1 Inhibiting DNA Polymerase Fights Cancer

      Drugs that inhibit DNA replication prevent cells dividing. Cytarabine (also known as cytosine arabinoside and arabinofuranosyl cytidine) is sold as Cytosar‐U, Tarabine PFS, Depocyt, and AraC. It inhibits eukaryote DNA polymerases α, δ, and ε, and is therefore widely used to treat cancers. It is on the World Health Organization's model list of essential medicines.

      Example 4.1 The Meselson and Stahl Experiment

      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 E. 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 halfway 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.

      DNA Synthesis Requires an RNA Primer

      DNA polymerase III cannot itself initiate the synthesis of DNA. The enzyme primase is needed to catalyze the formation of a short stretch of RNA complementary in sequence to the DNA template strand (Figure 4.1). This RNA chain, the primer, is needed to prime (or start) the synthesis of the new DNA strand. DNA polymerase III catalyzes the formation of a phosphodiester bond between the 3′‐hydroxyl group of the RNA primer and the 5′‐phosphate group of the appropriate deoxyribonucleotide. Several RNA primers are made along the length of the lagging strand template. Each is extended in the 5′ to 3′ direction by DNA polymerase III until it reaches the 5′ end of the next RNA primer. In prokaryotes the lagging strand is primed about every 1000 nucleotides whereas in eukaryotes this takes place every 200 nucleotides.

      RNA Primers Are Removed

      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′ to 3′ exonuclease activity and then uses its 5′ to 3′ polymerizing activity to incorporate deoxyribonucleotides. In this way, the entire RNA primer is replaced by DNA. Synthesis of the lagging strand is completed by the enzyme DNA ligase, which joins the DNA fragments together by catalyzing the formation of phosphodiester bonds between adjacent fragments.

      Eukaryotic organisms use an enzyme called ribonuclease H to remove their RNA primers. These enzymes break phosphodiester bonds in an RNA strand that is hydrogen‐bonded to a DNA strand. Within each cell, ribonuclease H2 is involved in removing RNA primers during replication of nuclear genomic DNA and ribonuclease H1 is involved in removing RNA primers during replication of mitochondrial genomic DNA.

      The Self‐Correcting DNA Polymerase

      The genome of E. coli consists of about 4.6 × 10⁶ base pairs of DNA. DNA polymerase III makes a mistake about every 1 in 10⁵ 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 a 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 (Figure 4.2b). 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 continues. DNA polymerase III hence functions as a self‐correcting enzyme.

      Mismatch Repair Backs Up the Proofreading Mechanism

The proofreading function of DNA polymerase III improves the accuracy of DNA replication about 100‐fold. However, sometimes the enzyme does miss a nucleotide that has been incorrectly inserted into the newly synthesized DNA strand. Cells have evolved a backup mechanism, mismatch repair, that detects when an incorrect nucleotide has been inserted into the daughter strand (Figure 4.3). The repair mechanism relies on the cell being able to distinguish, within the double helix, between the template strand (the parental strand) and the newly synthesized strand (the daughter strand).

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