Kinetics of DNA polymerase gamma upon mutation and nucleoside analog exposure

描述（由申请方提供）：线粒体毒性可能由人线粒体DNA聚合酶3（DNA pol 3）的活性改变引起，该酶负责线粒体基因组的复制。在影响约60，000名美国人的许多线粒体疾病（1）和目前影响估计一百万美国人的HIV中，已经观察到由DNA pol 3活性缺陷引起的线粒体DNA缺失或耗尽。DNA pol 3中的几个已知点突变与线粒体疾病有关，例如阿尔珀斯综合征，其特征在于剧烈癫痫发作和肝脏疾病，并导致儿童早期死亡，以及进行性眼外肌麻痹，一种成人发作的疾病，其导致上睑下垂和共济失调，通常是非致命的。已经努力确定通过突变改变DNA pol 3活性的分子机制，其可以源于催化循环中的任何步骤的修饰，包括核苷酸掺入效率、对引入的核苷酸和DNA模板的亲和力以及总体保真度。然而，大多数突变的动力学特征尚未进行。对于感染HIV的患者，核苷逆转录酶抑制剂（NRTI）是治疗的关键组成部分，但不幸的是，DNA pol 3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。可能导致类似于进行性眼外肌麻痹的症状，以及乳酸性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NRTI的需求，测试这些新药的DNA pol 3活性以评估安全性至关重要。该提案旨在表征四种DNA pol 3突变体A957 P、A957 S、R1096 H和R1096 C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Alpers综合征的严重程度的疾病相关。首先，生物物理方法将被用来表征突变酶的整体结构的变化，稳态和前稳态动力学将被用来确定毒性的分子机制。其次，DNA pol 3的预稳态动力学的阐明将用于测量新型抗HIV药物FLT的掺入效率和切除率，已知FLT表现出一定的毒性。确定突变和NRTI治疗如何改变DNA pol 3的动力学机制是理解线粒体疾病的原因和进展的关键一步，其中大多数目前无法治愈，并评估正在开发的NRTI药物的安全性。

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.