AZT-resistant mutants of DNA polymerase beta identified by in vivo selection: A structure-function study of polymerase substrate specificity.

摘要

Efficient and accurate synthesis of DNA requires that DNA polymerases maintain highly specific, yet flexible, interactions with nucleotide substrates. In order to investigate molecular mechanisms of polymerase substrate specificity, I developed an in vivo selection to identify mutant forms of the eukaryotic DNA repair enzyme DNA polymerase beta (Pol beta) that are resistant to the substrate analog AZT. The selection utilizes the ability of Pol beta to substitute for E. coli DNA polymerase I (Pol I). The SC18-12 strain carries a mutation that inactivates Pol I, inhibiting the growth of the cells. Expression of Pol beta complements the DNA replication defect of these cells, restoring their ability to grow. The Pol I-deficient cells are dependent on Pol beta activity, so inhibition of Pol beta by AZT is lethal. This allows selection of drug resistant Pol beta mutants, which can complement the Pol I defect in the presence of AZT.;I used this selection to screen a library of randomly mutated Pol beta molecules, and identified eleven AZT-resistant mutants. Each of these enzymes carried a single amino acid substitution, and the eleven changes were distributed among all domains of the Pol beta protein. None of the changes were in the immediate vicinity of the bound nucleotide substrate.;Three mutations occurred in a loop in the Pahn domain composed of residues 240--253. In vitro characterization of these three mutant enzymes revealed that two of the mutants, Arg253Met and Asp246Val, were less efficient than wildtype Pol beta at incorporation of AZT-TP into DNA, but were equally efficient at incorporation of dTTP, the natural substrate. These enzymes are the first polymerase mutants to display in vitro such a pronounced increase in AZT resistance compared to WT. The third enzyme, Glu249Lys, exhibited a mutator phenotype, attributable to its tendency to extend mispaired termini. The Glu249Lys enzyme exhibited a moderately increased binding affinity for nucleotides, while the Arg253Met mutant exhibited a substantially decreased binding affinity for dTTP. These results indicate that substrate specificity is influenced by residues distant from the nucleotide binding pocket, and suggest that the Palm domain loop participates in the binding of nucleotide substrates.