Nucleoside reverse transcriptase inhibitors (NRTIs) are an important class of
antiviral drugs used to manage
infections by human immunodeficiency virus, which causes
AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and
mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human
mitochondrial DNA polymerase has been established both in vitro and in vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family
DNA polymerases, which participate in DNA repair and DNA lesion bypass in vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human
DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5'-phosphorylated forms of
tenofovir,
lamivudine,
emtricitabine, and
zidovudine. For the six
enzymes, all of the
drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural
nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming
nucleotide analog. In general, the 5'-triphosphate forms of
lamivudine and
zidovudine were better substrates than
emtricitabine and
tenofovir for the six human
enzymes, although the substrate specificity profile depended on the
DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family
DNA polymerases is a potential mechanism of NRTI
drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.