DNA Polymerases Introduce Copying Errors and Also Correct Them

The first line of defense in preventing mutations is DNA polymerase itself. Occasionally, when replicative DNA polymerases (Pol δ and Pol ε) progress along the template DNA, an incorrect nucleotide is added to the growing 3′ end of the daughter strand. E. coli DNA polymerases, for instance, introduce about 1 incorrect nucleotide per 104 (ten thousand) polymerized nucleotides. Yet the measured mutation rate in bacterial cells is much lower: about 1 mistake in 109 (one billion) nucleotides incorporated into a growing strand. This remarkable accuracy is largely due to proofreading by E. coli DNA polymerases. Eukaryotic Pol δ and Pol ε employ a similar mechanism.

Proofreading depends on the 3′→5′ exonuclease activity of some DNA polymerases. When an incorrect base is incorporated during DNA synthesis, base pairing between the 3′ nucleotide of the nascent strand and the template strand does not occur. As a result, the polymerase pauses, then transfers the 3′ end of the growing chain to its exonuclease site, where the incorrect mispaired base is removed (Figure 5-33). Then the 3′ end is transferred back to the polymerase site, where this region is copied correctly. All three E. coli DNA polymerases have proofreading activity, as do the two eukaryotic DNA polymerases, δ and ε, used for replication of most chromosomal DNA in animal cells. It seems likely that proofreading is indispensable for all cells to avoid excessive mutations.

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FIGURE 5-33 Proofreading by DNA polymerase. All DNA polymerases have a similar three-dimensional structure, which resembles a half-opened right hand. The “fingers” bind the single-stranded segment of the template strand, and the polymerase catalytic activity (Pol) lies in the junction between the fingers and palm. As long as the correct nucleotides are added to the 3′ end of the growing strand, it remains in the polymerase site. Incorporation of an incorrect base at the 3′ end causes melting of the newly formed end of the duplex. As a result, the polymerase pauses, and the 3′ end of the growing strand is transferred to the 3′ →5′ exonuclease site (Exo) about 3 nm away, where the mispaired base, and probably other bases, are removed. Subsequently, the 3′ end flips back into the polymerase site and elongation resumes. See C. M. Joyce and T. T. Steitz, 1995, J. Bacteriol. 177:6321, and S. Bell and T. Baker, 1998, Cell 92:295.