FIDELITY OF DNA-SYNTHESIS CATALYZED BY HUMAN DNA-POLYMERASE ALPHA ANDHIV-1 REVERSE-TRANSCRIPTASE - EFFECT OF REACTION PH

The accuracy of DNA synthesis catalyzed by the Thermus aquaticus DNA p olymerase and the 3'-5' exonuclease-deficient Klenow fragment of Esche richia coli DNA polymerase I varies as a function of reaction pH (Ecke rt,K.A. and Kunkel,T.A. (1990) Nucleic Acids Res. 18, 3739-3744; Ecker t,K.A. and Kunkel,T.A. (1993) J. Biol. Chem. 268, 13462 - 13471). In t he current study, we demonstrate that the fidelity of human DNA polyme rase alpha increases 10-fold when the pH of the in vitro synthesis rea ction is lowered from pH 8.6 to pH 6.1 (37-degrees-C), as determined u sing a base substitution reversion assay to score polymerase errors wi thin the lacZalpha gene of bacteriophage M13mp2. Similarly, the base s ubstitution fidelity of DNA-dependent DNA synthesis by the human immun odeficiency virus type 1 reverse transcriptase (HIV-1 RT) was improved nine-fold at pH 6.5 relative to pH 8.0 (37-degrees-C). A detailed com parison of HIV-1 RT error specificity at neutral and low pH in a lacZa lpha forward mutation assay revealed that low pH suppresses both mispa iring-mediated and misalignment-mediated mutations; however, the chara cteristic HIV-1 RT pattern of mutational hotspots at homopolymeric seq uences is retained at the lower pH. Consistent with the presumption th at these mutations result, in part, from increased termination of DNA synthesis within the hotspot sequences relative to other homopolymeric sequences, the HIV-1 RT termination pattern during processive DNA syn thesis is not altered by low pH. The HIV-1 RT results are in agreement with our previous hypothesis that the observed increase in polymerase fidelity at low pH results from a decreased efficiency of continuing DNA synthesis from premutational DNA intermediates.