REDUCED FRAMESHIFT FIDELITY AND PROCESSIVITY OF HIV-1 REVERSE-TRANSCRIPTASE MUTANTS CONTAINING ALANINE SUBSTITUTIONS IN HELIX-H OF THE THUMB SUBDOMAIN

We have analyzed two human immunodeficiency virus (HIV-1) reverse tran scriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-pr imer. These enzymes, G262A and W266A, were previously shown to have gr eatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3'-azidodeoxythymidine 5'-trip hosphate. Here we describe their processivity and error specificity. T he results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence dependent termination of processive synthe sis and in error specificity, and (iii) the magnitude of the mutator e ffect relative to wild-type enzyme for deletions in homopolymeric sequ ences decreases as the length of the run increases. Thus amino acid su bstitutions in a subdomain thought to interact with the duplex templat e-primer confer a strand slippage mutator phenotype to a replicative D NA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active si te are critical determinants of processivity and fidelity. These effec ts, obtained in aqueous solution during catalytic cycling, are consist ent with and support the existing crystallographic structural model.