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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).

Figure 4.3. Mismatch repair of DNA.

We best understand this repair process in E. coli . The bacterium has an enzyme called Dam methylase that adds a ‐CH₃ group, called a methyl group, onto the A of the sequence 5′ GATC3′. This sequence occurs very frequently in DNA, about once every 256 bp. The methylation of DNA happens very soon after a DNA strand has been replicated. However, for a short time during replication the double‐stranded DNA molecule will have one strand methylated (the parental strand) and one strand not methylated (the daughter strand). The DNA molecule is said to be hemi‐methylated (half methylated). Because the newly synthesized strand has not yet been methylated the cell knows that if a mismatch in base pairing has occurred between the two strands it is the nonmethylated, newly synthesized, strand that must carry the mistake.

A protein called MutH binds on the newly synthesized strand at a site opposite a methylated A in the template strand. If there is no mismatched base pair nearby then MutH does nothing. However, if two other proteins called MutL and MutS have detected a mismatched base pair then MutH, which is an endonuclease, is activated and nicks (cleaves a phosphodiester bond between two nucleotides in) the unmethylated newly synthesized strand. This allows a stretch of DNA containing the mismatched base pair to be removed. Two different proteins are involved in removing the stretch of DNA. If MutH nicks the DNA 5′ to the mismatch (Figure 4.3a), then exonuclease VII degrades the DNA strand in the 5′ to 3′ direction. However, if MutH nicks the DNA 3′ to the mismatch (Figure 4.3b), then the DNA strand is removed by exonuclease I in the 3′ to 5′ direction. In either case, the gap in the daughter strand is then replaced by DNA polymerase III.