In Vitro Production and Screening of DNA Polymerase η Mutants for Catalytic Diversity

Abstract

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However, in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.