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

Abstract

We developed an in vivo selection to identify 3'-azido-3'-deoxythymidine (AZT)-resistant routants of rat DNA polymerase β (polβ). The selection utilizes pol β's ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol β allows SC18- 12 cells to grow, but they depend on pol β activity, so inhibition of pol β by AZT kills them. We screened a library of randomly mutated pol β 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 steadystate 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 β mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.