DNA polymerase active site is highly mutable: Evolutionary consequences

摘要

DNA polymerases contain active sites that are structurally super- imposable and highly conserved in sequence. To assess the signif- icance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquatiCus DNA polymerase l. After selection, by using genetic complementation, we obtained a library of approximately 8,000 active mutant DNA polymerases. of which 350 were se- quenced and analyzed. This is the largest collection of physiolog- ically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable whiIe preserving wild-type activity. A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative sub- stitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA poly- merase activity higher than the wiId-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.