Azole carboxamide nucleobases: Probes of DNA polymerase specificity and tools for mutagenesis
Survival of an organism is closely linked to the accuracy of DNA replication process. To examine the basis of DNA polymerase specificity, we have designed a set of azole carboxamide nucleobase analogs with promiscuous DNA polymerase incorporation properties. Analogs in this compound set displayed diversity in shape and electronic features. We took a steady state kinetics approach to study the DNA polymerases from different biological origins. Based on the results, a model was proposed for importance of shape and electronic features during base selection in the template strand. The model for template selection was further refined by studying the DNA polymerase reaction under pre-steady state conditions. Template incorporation studies with azole nucleobases were extended to the substrate incorporations and extensions reactions. Polymerases from varied biological origins showed substantial differences in their incorporation and extension properties. These distinctions among polymerases were further used to develop two distinct, tunable random mutagenesis methods. First, a model for mutagenesis using nucleobase analogs along the length of DNA sequence. Together, these methods represent a step further in generating and controlling sequence diversity using promiscuous nucleobase analogs.^
Vincent Jo Davisson, Purdue University, Donald E. Bergstrom, Purdue University.