Dynamic Characterization of Cancer-Associated Variants of DNA Polymerase Beta

描述(申请人提供)：癌症最终是由基因组的损伤引起的。DNA不断受到化学和环境的攻击，导致每个细胞每天大约有20,000个碱基对受损。如果不进行修复，这些受损的位置可能会在复制时导致失配诱导的突变，最终导致癌症。幸运的是，细胞拥有检测、移除和替换此类损伤的高效过程。这一过程中的关键酶之一是DNA聚合酶β(POLβ)，它负责确保保真度的步骤，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。因此，与“正常”或野生型(WT)POLβ相比，保真度或活性降低的POLβ突变体会导致癌症。虽然许多这样的突变体已经被生化表征，但WT Polβ的微小结构变化如何能够显著改变Polβ的功能仍不清楚。在DNA修复过程中，POLβ经历了一系列大规模、可逆的构象变化。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，即使在没有底物的情况下，WT Polβ在催化途径的所有阶段都会自行经历快速、可逆的构象变化。令人惊讶的是，这些动态运动似乎是催化途径后期阶段形成的构象样本。我们假设，在WT Pol beta中观察到的动态行为在癌症相关变体中发生了改变。本项目的目标是通过使用溶液状态松弛弥散核磁共振技术表征致突变POLβ变体的动力学来验证这一假设，并将这些研究的结果与WT POLβ的动力学以及先前表征每个变体诱导的突变种类的研究进行比较。 在超过30%的人类癌细胞中发现了波尔贝塔的突变形式。与WT Pol beta相比，这些突变体中的许多都降低了保真度或活性。这项研究将有助于解释POLβ基因的点突变如何导致突变变体。

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.