Infidelity of DNA synthesis by temperature-sensitive DNA polymerases from RNA tumor viruses

Abstract

An analysis of the role of reverse transcriptase in base-selection has been carried out using mutant DNA polymerases from RNA tumor viruses. Three temperature-sensitive DNA polymerases from avian sarcoma viruses and a wild type DNA polymerase from an avian leukosis virus were assayed for the accuracy with which they copy homopolymers in vitro. With poly(A) · oligo(dT) as a template, the frequency of incorporation of the non-complementary nucleotide, dCMP, into the poly(dT) product varied from 1 in 235 to 1 in 276. With poly(C) · oligo(dG) as template, the incorporation of dAMP into poly(dG) varied from 1 in 875 to approx. 1 in 1700. Analyses of the polynucleotide products of the reaction by equilibrium density centrifugation and digestion with snake venom phophodiesterase indicate that the non-complementary nucleotides are incorporated in phosphodiester linkage and are distributed throughout the length of the product. With poly(A) and poly(C) templates, the rate of incorporation of both complementary and non-complementary nucleotides by each mutant DNA polymerase was temperature sensitive compared to that of wild type DNA polymerase. These data demonstrate that the polymerase, itself, catalyses the incorporation of both complementary and non-complementary nucleotides. Partial heat denaturation of the mutant polymerases did not change the fidelity of copying poly(A). With poly(C) as template, however, partial heat inactivation changed the error frequency of two of the mutant polymerases. These results suggest that the mutattions studied may alter base discrimination by the polymerase in a template-dependent manner.