Exploring the Roles of Nucleobase Desolvation and Shape Complementarity during the Misreplication of O6-Methylguanine

O6-methylguanine (O6-MeG) is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles of hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and nonnatural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O6-MeG. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymeraseʹs active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O6-MeG observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of nonnatural nucleotides identified an analog that displays high selectivity for incorporation opposite O6-MeG compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis.