Although
antiviral nucleoside analog therapy successfully delays progression of
HIV infection to
AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial
polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating
antiviral nucleotide analogs into
DNA. Here, we explore the role of three highly conserved
amino acid residues in the active site of human pol gamma that modulate selection of
nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family
A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural
nucleotides and the five
antiviral nucleoside analogs currently approved for
antiviral therapy: AZT, ddC,
D4T, 3TC and
carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and
antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate
dideoxynucleotides and
D4T-MP. Mutation of Tyr951 to Phe renders the
enzyme resistant to
dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal
nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease
progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate
D4T-MP and
carbovir. Alteration of Glu895 to Ala slightly increased discrimination against
dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain
nucleotide analogs are discussed.