Kinetics of DNA polymerase gamma upon mutation and nucleoside analog exposure

描述(申请人提供)：线粒体毒性可由人类线粒体DNA聚合酶3(DNA Pol3)的活性改变引起，该酶负责复制线粒体基因组。在许多线粒体疾病中都观察到了由于DNA pol3活性不足而导致的线粒体DNA缺失或耗尽，这些疾病影响了大约60,000美国人(1)，以及目前影响大约100万美国人的艾滋病毒。DNA pol3的几个已知的点突变与线粒体疾病有关，例如阿尔珀斯综合征，其特征是剧烈癫痫和肝脏疾病，并导致儿童早期死亡，以及进行性外眼肌麻痹，这是一种成人发病的疾病，会导致上睑下垂和共济失调，通常不是致命的。人们已经努力确定DNA Pol3活性因突变而改变的分子机制，这可能源于催化循环中的任何步骤的改变，包括核苷酸掺入效率、与传入核苷酸和DNA模板的亲和力以及整体保真度。然而，对于大多数突变，还没有进行动力学表征。对于感染HIV的患者，核苷逆转录酶抑制剂(NRTI)是治疗的关键成分，但不幸的是，DNA Pol3也可以使用这些核苷类似物作为底物，导致线粒体DNA复制过程中的链终止。症状类似于进行性眼外肌麻痹，以及乳酸酸中毒和脂肪营养不良，毒性可能严重到需要停止这种类型的治疗。毒性和耐药性问题突出了对新的NRTI的需求，测试这些新药的DNA Pol3活性以评估安全性至关重要。这项建议旨在表征四个DNA pol3突变体A957P、A957S、R1096H和R1096C的毒性动力学机制，这些突变体与从轻度进行性眼外肌麻痹到阿尔珀斯综合征等疾病的严重程度有关。首先，将使用生物物理方法来表征突变酶的整体结构的变化，并将利用稳态和稳态前动力学来确定毒性的分子机制。其次，阐明DNA pol3的稳态前动力学将用于测量新型抗HIV药物Flt的掺入效率和切除率，已知该药物具有一定的毒性。确定突变和NRTI治疗如何改变DNA Pol3的动力学机制是了解线粒体疾病的原因和进展的关键一步，目前大多数线粒体疾病还没有治愈方法，也是评估正在开发的NRTI药物的安全性的关键一步。

DESCRIPTION (provided by applicant): 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.