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

We developed an in vivo selection to identify 3-'azido-3'-deoxythymidine (A ZT)-resistant mutants of rat DNA polymerase beta (pol beta). The selection utilizes pol beta's ability to substitute for Escherichia coli DNA polymera se I (pol I) in the SC18-12 strain, which lacks active pol I. pol beta allo ws SC18-12 cells to grow, but they depend on pol beta activity, so inhibiti on 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 A ZT, and identified AZT-resistant mutants. We purified two enzymes with nonc onservative mutations in the palm domain of the polymerase, The substitutio ns D246V and R253M result in reductions in the steady-state catalytic effic iency (K-cat/K-m) of AZT-TP incorporation. The efficiency of dTTP incorpora tion was unchanged for the D246V enzyme, indicating that the substantial de crease 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 locat ions of the Asp-246 and Arg-253 side chains indicate that substrate specifi city is influenced by residues distant from the nucleotide-binding pocket.