Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast

Abstract

DNA-directed DNA polymerases have been broadly classified into seven families based on their sequence homology (1). It is surprising to learn that enzymes such as DNA polymerases, which carry out pivotal role during DNA replication, repair, and recombination, are poorly conserved amongst different families, but within a given family, all the members are highly conserved. These observations have profound implications and suggest that DNA polymerases have been plastic during evolution, but can tolerate multiple mutations (2). The mutability of DNA polymerases has been utilized extensively in our studies and has shed light on structure-function relationships of each domain. Any single amino acid residue or the entire domain can be randomly mutagenized and the active mutants can be selected by genetic complementation. Here we describe the complementation of Saccharomyces cerevisiae Pol3 (Pol δ) by utilizing a common technique in yeast genetics known as “plasmid shuffling,” where the wild-type copy of the Pol3 present in a Ura3 selective marker plasmid is exchanged or genetically complemented for in vitro mutated version(s) of Pol3 in the domain-of-interest. Since Pol3p is essential for viability of yeast, only those mutants that genetically complement the loss of wild-type Pol3p survive.