Selection and characterization of AZT-resistant mutants of DNA Polymerase beta (Meeting abstract).

Important note: Information in this article was accurate in 1998. The state of the art may have changed since the publication date.

Selection and characterization of AZT-resistant mutants of DNA Polymerase beta (Meeting abstract).

Proc Annu Meet Am Assoc Cancer Res; 38:A1216 1997. Unique Identifier : AIDSLINE ICDB/98638216
Kosa JL; Sweasy JB; Yale Medical School, New Haven, CT 06520

Abstract: DNA polymerase beta (Pol beta) is implicated in DNA repair synthesis, a role consistent with its in vitro aptitude for filling short gaps in DNA. Pol beta is inhibited in vitro by the nucleoside analog AZT, which Pol beta incorporates into DNA, resulting in chain termination. We developed an in vivo selection scheme to identify mutant forms of Pol beta which are resistant to the drug AZT. The selection utilizes the ability of Pol beta to substitute for the E.coli polymerase Pol l. The recA718polA12 strain can not grow at 37 C due to a temperature sensitive mutation inactivating DNA polymerase I (Pol Its). Expression of Pol beta complements the replication defect of these cells, restoring growth. The Pol Its cells are dependent on Pol beta activity, so inhibition of Pol beta by AZT is lethal. This allows us to select Pol beta mutants which are able to complement the Pol Its phenotype in the presence of AZT from an expression library of randomly mutated Pol beta. We have identified clones which grow in the presence of AZT. The phenotypes of the mutants vary qualitatively. Each mutant is altered in a different sector of the enzyme. The mutations may affect substrate specificity by altering the dNTP binding pocket, the positioning of the primer-template DNA, or the conformational flexibility of the enzyme. Biochemical characterization of these drug resistant mutants should provide insight into the molecular interactions of Pol beta with nucleotide substrates.

Keywords: Cell Division/GENETICS Cloning, Molecular DNA Polymerase beta/*GENETICS/METABOLISM DNA Repair Genetic Complementation Test Phenotype Zidovudine/*PHARMACOLOGY ABSTRACT
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