3′-azido-3′-deoxythymidine-resistant mutants of DNA polymerase β identified by in vivo selection

We developed an in vivo selection to identify 3-'azido-3'-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase beta (pol beta). The selection utilizes pol beta's ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol beta allows SC18-12 cells to grow, but they depend on pol beta activity, so inhibition of pol beta by AZT kills them. We screened a library of randomly mutated pol beta cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase, The substitutions D246V and R253M result in reductions in the steady-state catalytic efficiency (K-cat/K-m) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance, The R253M enzyme exhibits significantly higher K-m(dTTP) and K-cat(dTTP) values, implying that the incorporation reaction is altered. These are the first pol beta mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.