AZT-resistant mutants of DNA polymerase βidentified by in vivo selection

The DNA repair enzyme DNA polymerase β(Pol β) is inhibited by the substrate analog AZT. We developed a selection scheme to identify mutant forms of Pol βwhich are resistant to AZT. The selection utilizes the ability of Pol β to replace the. K. coli polymerase Pol I. The recA718 polA12 strain, which carries a temperature sensitive mutation inactivating Pol I (Pol A-), is unable to grow on rich medium at 37°C. Expression of Pol βallows these cells to grow at 37T. The Pol A- cells are dependent on Pol β, so inhibition of Pol βby AZT is lethal. This allows us to select drug resistant Pol βmutants, which are able to complement the Pol A- phenotype in the presence of AZT.We have used this selection to screen a library of randomly mutated Pol β. and have identified AZT-resistant mutant enzymes. We have purified two of these enzymes, and found that they are as active as wildtype Pol βwith dNTP substrates, but exhibit a dramatic inability to incorporate AZT-TP. We found that the substitutions D246V and R253M result in 10-fold and 500-fold reductions in the efficiency of AZT-TP incorporation. The pffect was primarily due to a large decrease in Kcat for AZT-TP incorporation, suggesting that these mutations disrupt the chemical step of the reaction. This is consistent with the idea that these mutations perturb the structure of the Palm domain, altering active site geometry in a way that increases discrimination against AZT-TP.These findings demonstrate that this genetic screen is effective for identifying drug resistant inutant.s of Pol β. Study of the molecular basis of these mutants' AZT resistance will providn insigM into thp interactions of Pol βwith nucleotido substrates.