DNA Replication:- Replication Is Very Accurate
Replication proceeds with an extraordinary degree of fidelity. In E. coli, a mistake is made only once for every 109 to 1010 nucleotides added. For the E. coli chromo some of ~4.6 106 bp, this means that an error occurs only once per 1,000 to 10,000 replications. During poly merization, discrimination between correct and incorrect nucleotides relies not just on the hydrogen bonds that specify the correct pairing between complementary bases but also on the common geometry of the standard A=T and G≡C base pairs (Fig. 1). The active site of DNA polymerase I accommodates only base pairs with this geometry. An incorrect nucleotide may be able to hydrogen-bond with a base in the template, but it generally will not fit into the active site. Incorrect bases can be rejected before the phosphodiester bond is formed. The accuracy of the polymerization reaction itself, however, is insufficient to account for the high degree of fidelity in replication. Careful measurements in vitro have shown that DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct ones. These mistakes sometimes occur because a base is briefly in an unusual tautomeric form , allowing it to hydrogen-bond with an incorrect partner. In vivo, the error rate is reduced by additional enzymatic mechanisms. One mechanism intrinsic to virtually all DNA polymerases is a separate 3n5 exonuclease activity that double-checks each nucleotide after it is added. This nuclease activity permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base pairs (Fig. 2). If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited. This kinetic pause provides the opportunity for a correction. The 3→5 exonuclease activity removes the mispaired nucleotide, and the polymerase begins again. This activity, known as proofreading, is not simply the reverse of the polymerization reaction (Eqn 25–1), because pyrophosphate is not involved. The polymerizing and proofreading activities of a DNA polymerase can be measured separately. Proofreading improves the inherent accuracy of the polymerization re action 102- to 103-fold. In the monomeric DNA polymerase, I, the polymerizing and proofreading activities have separate active sites within the same polypeptide. When base selection and proofreading are combined, DNA polymerase leaves behind one net error for every 106 to 108 bases added. Yet the measured accuracy of replication in E. coli is higher still. The additional accuracy is provided by a separate enzyme system that repairs the mismatched base pairs remaining after replication. We describe this mismatch repair, along with other DNA repair processes, in Section 25.2.
FIGURE 1 Contribution of base-pair geometry to the fidelity of DNA replication. (a)The standard A=T and G≡C base pairs have very similar geometries, and an active site sized to fit one (blue shading) will generally accommodate the other. (b) The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase I.
FIGURE 2 An example of error correction by the 3→5 exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease activity ahead of the polymerase activity as the en zyme is oriented in its movement along the DNA. A mismatched base (here, a C–A mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 3→5 exonuclease activity, then resumes its polymerase activity in the 5→3 direction.
