The role of the human mitochondrial polymerase in the toxicity of nucleoside analogs and aging
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The toxic side effects associated with the administration of nucleoside analogs used to treat HIV are correlated with the kinetics of incorporation by the human mitochondrial polymerase γ (Pol γ). The reconstitution of recombinant human enzyme has allowed for a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. One nucleoside analog in particular, zidovudine (AZT), exhibits unique kinetics of incorporation by Pol γ. Evidence is presented supporting a model in which the kinetics of incorporation of AZT differ from that of natural nucleotides and other nucleoside analogs, in that phosphoryl transfer is reversibly linked to binding by way of a slower than normal enzyme isomerization following phosphoryl transfer. The implication of the unique reaction kinetics on the toxicity associated with the use of AZT is also discussed. The most toxic nucleoside analog approved for treatment of HIV is zalcitabine (ddC). It was shown previously that the incorporation of ddC by Pol γ is relatively efficient and that the removal by the proofreading exonuclease was too slow to measure under the conditions used. Both efficient incorporation and slow removal are likely to contribute to the observed clinical toxicity. I revisited this phenomenon by measuring the rate of excision more accurately and under various conditions in order to better understand the mechanistic basis for the slow removal of ddC. In addition to a role in the toxicity of nucleoside analogs used to treat HIV, Pol γ may play an important part in the process of aging. The mitochondrial theory of aging states that electrons derived from the electron transport chain during normal respiration produce especially high levels of reactive oxygen species (ROS) in the mitochondria. These ROS can damage the mitochondrial genome, compromising its integrity. One of the most common products of oxidative damage to DNA is 8-oxodG. I have examined the kinetic parameters governing the replication of oxidatively damaged DNA by Pol γ and show that replication fidelity is reduced when incorporation is performed when 8-oxodG is the templating base. I provide evidence suggesting that oxidized free nucleotide (8-oxodGTP) may also be mutagenic.