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The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.

Polynucleotide templates were copied by avian myeloblastosis virus DNA polymerase ("reverse transcriptase") and the frequency and distribution of errors were determined. The error rate with [r(pA)2500-d(pT)12-18] template-initiator under a variety of conditions was approximately 1/600, i.e. one incorrect dCMP incorporated for 600 correct dTMP polymerized. Addition of the metal chelator o-phenanthroline to the reaction inhibited the incorporation of correct and incorrect nucleotides proportionately. The enzyme inhibited a pH optimum of 8.5 and the error rate remained constant over a range of pH (6.0 to 10.0). The rate of polymerization was greater at higher temperatures and approximately doubled for every 10 degrees increase. The error rate was constant at all temperatures. These results indicate that the purified avian myeloblastosis virus DNA polymerase synthesizes polydeoxynucleotides with an unusually large number of errors in base-pairing. Velocity sedimentation of the products of the reaction obtained at various times during the course of synthesis indicate that: (a) the entire length of the 14 S template was copied, and (b) the incorporation of the incorrect nucleotide did not terminate chain propagation. Isopyknic banding in neutral and alkaline cesium sulfate gradients showed that the noncomplementary nucleotides are incorporated into the polydeoxynucleotide product. Stepwise degradation and nearest neighbor analysis of the reaction product indicated that (a) the correct and incorrect nucleotides are present in phosphodiester linkages, (b) the errors are not concentrated at either termini; and (c) the errors are uniformly distributed throughout the newly synthesized polydeoxynucleotide.

Homogeneous DNA polymerase ("reverse transcriptase") from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity. The substrates were defined template-initiator complexes in which different radioactive nucleotides were present at the 3'-OH termini of the initiator. Even when the number of molecules of enzyme was equal to the number of initiator termini there was no significant release of radioactivity with any of the template-initiator combinations tested. Under similar conditions, the nuclease activity associated with either Escherichia coli or T4DNA polymerases rendered more than 90% of the initiator termini acid-soluble. The ratio of exodeoxyribonuclease activity to protein with avian myeoblastosis DNA polymerase is less than 0.003% of that obtained with E. coli DNA polymerase I. Furthermore, avian myeloblastosis virus DNA polymerase failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.