Mitochondrial toxicity can result from altered activity of human mitochondrial DNA polymerase 3 (DNA pol 3), the enzyme responsible for replication of the mitochondrial genome. Mitochondrial DNA deletion or depletion resulting from deficient DNA pol 3 activity has been observed in many mitochondrial diseases which affect about 60,000 Americans (1), and in HIV which currently affects an estimated one million Americans. Several known point mutations in DNA pol 3 have been linked to mitochondrial diseases such as Alpers Syndrome, which is characterized by violent seizures and liver disease and causes death in early childhod, and progressive external ophthalmoplegia, an adult onset disorder which causes ptosis and ataxia and is usualy non-fatal. Effort has been made to determine the molecular mechanism of the alteration of DNA pol 3 activity by mutation, which can stem from the modification of any of the steps in the catalytic cycle including the nucleotide incorporation efficiency, the affinity for incoming nucleotides and the DNA template, and the overall fidelity. However, kinetic characterization has not been undertaken for most mutations. For patients infected with HIV, nucleoside reverse transcriptase inhibitors (NRTIs) are a critical component of treatment, but unfortunately DNA pol 3 also can use these nucleoside analogs as substrates, resulting in chain termination during mitochondrial DNA replication. Symptoms similar to those seen in progressive external ophthalmoplegia can result, as well as lactic acidosis and lipodystrophy, and toxicity can be severe enough to require the halt of this type of treatment. Problems of toxicity and resistance highlight the demand for new NRTIs, and it is critical to test these novel drugs for DNA pol 3 activity to assess safety. This proposal seeks to characterize the kinetic mechanism of toxicity for four DNA pol 3 mutants, A957P, A957S, R1096H, and R1096C, which are associated with disease ranging in severity from mild progressive external ophthalmoplegia to Alpers Syndrome. First, biophysical methods will be used to characterize changes in the overall structure of the mutant enzymes, and steady-state and pre-steady-state kinetics will be utilized to determine the molecular mechanism of toxicity. Second, elucidation of the pre-steady-state kinetics of DNA pol 3 will be used measure the efficiency of incorporation and the rate of excision of the novel anti-HIV drug, FLT, which is known to exhibit some toxicity. Determining the kinetic mechanism of how mutation and NRTI treatment alters DNA pol 3 is a critical step in understanding the cause and progression of mitochondrial diseases, most of which currently have no cure, and for assessing the safety of NRTI drugs under development.
Diseases related to mitochondrial DNA depletion and deletion can stem from natural mutation of human mitochondrial DNA polymerase 3 or from inhibition of this enzyme by nucleoside analog drugs which are vital for treatment of HIV infection. This proposal aims to use enzyme kinetics to determine how DNA polymerase 3 mutations associated with Alpers Syndrome and progressive external ophthalmoplegia affect the catalytic activity, and to characterize DNA polymerase 3 inhibition upon treatment with a novel nucleoside analog drug. Determining these molecular mechanisms of toxicity is critical for understanding the cause and progression of these currently incurable mitochondrial disorders, and for assessing the safety of anti-HIV drugs currently under development.
