Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma

Mitochondrial toxicity can result hom antiviral nucleotide analog therapy u sed to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochond rial DNA polymerase (pol gamma) by comparing the insertion and exonucleolyt ic removal of six antiviral nucleotide analogs. Apparent steady-state K-m a nd k(cat) values for insertion of 2 ' ,3 ' -dideoxy-TTP (ddTTP), 3 ' -azido -TTP (AZT-TP), 2 ' ,3 ' -dideoxy-TTP (dd-TTP), 2 ' ,3 ' -didehydro-TTP (D4T -TP), (-)-2 ' ,3 ' -dideoxy-3 ' -thiacytidine (3TC-TP), and carbocyclic 2 ' ,3 ' -didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utili zed by pol gamma in vitro as efficiently as natural deoxynucleotides, where as AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain el ongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV fro m DNA predicts persistence in vivo following successful incorporation. In c ontrast, removal of 3'-terminal 3TC residues was 50% as efficient as natura l 3 ' termini. Finally, me observed inhibition of exonuclease activity by c oncentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termi nation, persistence of these analogs in DNA and inhibition of exonucleolyti c proofreading may also contribute to mitochondrial toxicity.