MISPAIR EXTENSION FIDELITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE TRANSCRIPTASES WITH AMINO-ACID SUBSTITUTIONS AFFECTING TYR115

The role of Tyr115 of human immunodeficiency virus type 1 reverse tran scriptase (HIV-1 RT) in the mispair extension fidelity of DNA dependen t DNA synthesis was analysed by using a series of 15 mutant enzymes wi th substitutions at Tyr115. Their kinetic parameters for elongation us ing homopolymeric RNA-DNA and heteropolymeric DNA-DNA complexes showed major effects of the amino acid substitutions on the K-m value for dN TP. Enzymes with large hydrophobic residues at position 115 displayed lower K-m values than enzymes with small and charged amino acids at th is position. The influence of all these amino acid replacements in mis pair extension fidelity assays was analyzed using three different mism atches (A:C, A:G and A:A) at the 3'-terminal position of the primer DN A. For the A:C mispair, a 2.6-33.4-fold increase in mispair extension efficiency (f(ext)) was observed as compared with the wild-type enzyme . Unexpectedly, all the mutants tested as well as the wild-type RT wer e very efficient in extending the A:G and A:A transversion mispairs. T his effect was due to the template-primer sequence context and not to the buffer conditions of the assay. The data support a role of Tyr115 in accommodating the complementary nucleotide into the nascent DNA whi le polymerization takes place.