The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucle otide is covalently added to the growing chain. DNA polymerase enzymes are unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH ter minus that can prime DNA synthesis. In this way, DNA polymerase functions as a attached to DNA polymerase “self-correcting” enzyme that removes its own polymerization errors as it moves chews back to create a base- along the DNA (Figure 5–8 and Figure 5–9).
paired 3˜-OH end on the primer
The self-correcting properties of the DNA polymerase depend on its require ment for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer. By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism: errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer.
DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand
Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNA replication. In this example, a C is accidentally incorporated at the growing 3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes the misincorporated nucleotide is a specialized member of a large class of enzymes, known as exonucleases, that cleave nucleotides one at a time from the ends of polynucleotides.
Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.
There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1).
Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction
The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.
Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another