Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity

Abstract

DNA replication, transcription, and translation rely on the strict Watson-Crick base pairing rules to ensure faithful transmission of genetic information. The Watson-Crick pairing rules are determined by the predominant neutral tautomeric forms of the nucleic acid bases. Incorrect base pairing during replication, if left unrepaired, leads to transition or transversion point mutations. Spontaneous mutagenesis from replication errors is believed to be a prominent source of base substitution errors in tumor suppressor genes in multiple forms of cancer. Rare tautomeric and ionized nucleotide bases can form mismatches that conform to the Watson-Crick like geometry, subverting proof reading mechanisms. These tautomeric and anionic mismatches have long been suspected to contribute to spontaneous replication errors; they have proved difficult to visualize as the conformational changes are subtle and involve the rearmament of protons. Nuclear magnetic resonance relaxation dispersion techniques have allowed for the characterization of a highly sequence-dependent kinetic network connecting the wobble dG·dT mismatch to multiple Watson-Crick-like tautomeric and anionic dG·dT mismatch ‘excited states’. We have obtained evidence in support of a kinetic model for misincorporation which introduces a rate-limiting on-pathway tautomerization or ionization step that leads to Watson-Crick-like mismatches prior to incorporation through the canonical synthesis pathway. This kinetic model can account for i) the three orders of magnitude difference seen in vitro between rates of correct and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low as 10−6, and iii) the poorly understood sequence dependence of polymerization errors.