A replication fork therefore has an asymmetric structure (Figure 5–7). The DNA daughter strand that is synthesized continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand. For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ type of DNA polymerase.
The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms
As discussed above, the fidelity of copying DNA during replication is such that only about one mistake occurs for every 1010 nucleotides copied. This fidelity is much higher than one would expect from the accuracy of complementary base-pairing. The standard complementary base pairs (see Figure 4–4) are not the only ones possible. For example, with small changes in helix geometry, two hydrogen bonds can form between G and T in DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispair without a change in helix geometry: the rare tautomeric form of C pairs with A instead of G, for example.
If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on the initial base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairings that might have occurred.
Figure 5–6 Two replication forks moving in opposite directions on a circular chromosome. An active zone of DNA replication moves progressively along a replicating DNA molecule, creating a Y-shaped DNA structure known as a replication fork: the two arms of each Y are the two daughter DNA molecules, and the stem of the Y is the parental DNA helix. In this diagram, parental strands are orange; newly synthesized strands are red. 1 µm (Micrograph courtesy of Jerome Vinograd.)
Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesized DNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis. Because both daughter DNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments, named after the scientist who discovered them. Right, the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the most recently made.
DNA polymerase performs the first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the grow-AAAAAAAAA ing chain, the enzyme must undergo a conformational change in which its “grip” tightens around the active site (see Figure 5–4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.