Dynamic Characterization of Cancer-Associated Variants of DNA Polymerase Beta

DNA 聚合酶 Beta 癌症相关变体的动态表征

• 批准号: 8457702
• 负责人: Elizabeth Margaret Moscato-Goodpaster
• 金额: $ 5.22万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8457702
• 关键词: Adopted; Affinity; Base Excision Repairs; Base Pairing; Behavior; Binding; Cancer Etiology; Cells; Chemistry; Complex; DNA; DNA Damage; DNA Polymerase beta; DNA Repair; DNA lesion; DNA-Directed DNA Polymerase; Ensure; Enzymes; Equilibrium; Excision; Exhibits; Genome; Genomics; Goals; Human; Induced Mutation; Kinetics; Left; Lesion; Link; Malignant Neoplasms; Mammalian Cell; Minor; Molecular Conformation; Molecular Models; Motion; Mutate; Mutation; Nucleotides; Pathway interactions; Point Mutation; Polymerase; Process; Relative (related person); Relaxation; Role; Sampling; Series; Site; Solutions; Sorting - Cell Movement; Staging; Techniques; Testing; Variant; assault; base; cancer cell; deviant; environmental chemical; in vitro Bioassay; in vivo; metaplastic cell transformation; models and simulation; molecular modeling; mutant; public health relevance; repaired; research study

DESCRIPTION (provided by applicant): Cancer is ultimately caused by damage to the genome. DNA is constantly subjected to chemical and environmental assaults, resulting in approximately 20,000 damaged base pairs per cell per day. Left unrepaired, these lesioned sites could result in mismatched-induced mutations upon replication, eventually causing cancer. Fortunately, cells possess highly effective processes which detect, remove, and replace such lesions. One of the crucial enzymes in this process is DNA Polymerase beta (Pol beta), which is responsible for the fidelity-ensuring step of inserting the appropriate nucleotide into a gap generated by prior excision of a damaged base pair. Accordingly, Pol beta mutants with reduced fidelity or activity relative to "normal" or wild-type (WT) Pol beta cause cancer. Although many such mutants have been biochemically characterized, the ways in which minor structural changes to WT Pol beta can dramatically alter Pol beta's function still remain unclear. During the process of DNA repair, Pol beta undergoes a series of large-scale, reversible conformational changes. These changes have traditionally been assumed to occur only in the presence of substrate. Recently, however, we and others have found that, along all stages of the catalytic pathway, WT Pol beta undergoes rapid, reversible conformational changes on its own, even in the absence of substrate. Surprisingly, these dynamic motions appear to sample conformations formed at later stages of the catalytic pathway. We hypothesize that the dynamic behaviors observed in WT Pol beta are altered in cancer-associated variants. This goal of this project is to test this hypothesis by characterizing the dynamics of mutagenic Pol beta variants using solution-state relaxation dispersion NMR techniques and to compare the results of these studies with both the dynamics of WT Pol beta and with previous studies which have characterized the sorts of mutations each variant induces. Mutant forms of Pol beta have been found in over 30% of all human cancer cells. Many of these mutants have reduced fidelity or activity relative to WT Pol beta. This study will help explain how point mutations in Pol beta can result in mutagenic variants.

描述（由申请人提供）：癌症最终是由基因组损伤引起的。DNA不断受到化学和环境的攻击，导致每个细胞每天大约20，000个受损碱基对。如果不修复，这些损伤部位可能会在复制时导致错配诱导突变，最终导致癌症。幸运的是，细胞具有高度有效的过程，可以检测、去除和替换这些病变。这个过程中的关键酶之一是DNA聚合酶β（Pol β），它负责将适当的核苷酸插入到先前切除受损碱基对产生的缺口中的确保完整性的步骤。因此，相对于“正常”或野生型（WT）Pol β具有降低的保真度或活性的Pol β突变体引起癌症。尽管许多这样的突变体已经被生化表征，但WT Pol β的微小结构变化可以显著改变Pol β的功能的方式仍然不清楚。在DNA修复过程中，Pol beta经历了一系列大规模的可逆构象变化。这些变化传统上被认为只在底物存在的情况下发生。然而，最近，我们和其他人发现，沿着催化途径的所有阶段，WT Pol β自身经历快速、可逆的构象变化，即使在没有底物的情况下也是如此。令人惊讶的是，这些动态运动似乎是在催化途径的后期阶段形成的样品构象。 我们假设在WT Pol β中观察到的动态行为在癌症相关变体中改变。本项目的目标是通过使用溶液状态弛豫分散NMR技术表征诱变Pol β变体的动力学来验证这一假设，并将这些研究的结果与WT Pol β的动力学以及先前的研究进行比较，这些研究表征了每个变体诱导的突变种类。 Pol β的突变形式已在超过30%的人类癌细胞中发现。这些突变体中的许多相对于WT Pol β具有降低的保真度或活性。这项研究将有助于解释Pol β的点突变如何导致诱变变体。