DNA polymerase is the enzyme responsible for the addition of nucleotide bases to the growing strand during DNA replication. Since the nucleotide sequence of a genome determines the development and function of a given organism, it is essential to synthesize accurate replicas of the existing genome during DNA replication. Typically, DNA polymerases maintain high fidelity durng DNA replication, incorporating only a single mismatched nucleotide for every 109 nucleotides added. Thus, if a mismatch occurs between nitrogen-containing bases in addition to the standard complementary base pair, the DNA polymerase adds that nucleotide to the growing strand, resulting in frequent mutations. Errors in DNA replication are corrected by two mechanisms, called proofreading and strand-directed mismatch repair.
Proofreading refers to the initial mechanism of correcting mismatched base pairs from a growing DNA strand, and it is performed by DNA polymerase. DNA polymerase was proofread in two steps. The first proofreading occurs before the addition of new nucleotides to the growing strand. The affinity of correct nucleotides for DNA polymerase is many times higher than that of incorrect nucleotides. However, after the incoming nucleotide is bound to the template by hydrogen bonding, but before the nucleotide is bound to the growing strand by the action of DNA polymerase, the enzyme should undergo a conformational change. During the conformational change of DNA polymerase, faulty base pairs of nucleotides are prone to dissociation from the template. Thus, this step allows the DNA polymerase to "scrutinize" nucleotides before adding them permanently to the growing strand.
proofreading
The second proofreading step is called nucleoside proofreading. In rare cases, it occurs immediately after incorporation of a mismatched nucleotide into the growing strand. The DNA polymerase was unable to add a second nucleotide next to the mismatched nucleotide. The separate catalytic sites of DNA polymerase, called 3 'to 5' proofreading exonucleases, digest mismatched nucleotides from the growing strand.
Strand-directed mismatch repair
Despite the proofreading mechanism, DNA polymerase may still incorporate incorrect nucleotides into the growing strand during DNA replication. Replication errors that escape from proofreading are removed by strand-directed mismatch repair. This system detects the distortion potential in the DNA helix due to base pair mismatch. However, the repair system should identify the incorrect base from the existing base before replacing the mismatched base. Typically, Escherichia coli relies on the DNA methylation system to recognize old DNA strands in the double helix, since newly synthesized strands may not undergo DNA methylation quickly. In Escherichia coli, the A residue of GATC is methylated. The fidelity of DNA replication is increased 102-fold due to the strand-directed mismatch repair system.
In strand-directed mismatch repair, three complex proteins cross a newly synthesized strand of DNA. The first protein, called MutS, detects and binds to distortions in the DNA double helix. A second protein, called MutL, detects and binds to MutS, attracting a third protein, called MutH, for distinguishing unmethylated or newly synthesized chains. Upon binding, MutH cleaves the unmethylated DNA strand upstream of the G residue in the GATC sequence. Exonucleases are responsible for degradation of the downstream strand to the mismatch. However, this system degrades regions of less than 10 nucleotides, which are readily de novo synthesized by DNA polymerase 1. The eukaryotic Mut protein is homologous to the Escherichia coli Mut protein.
