Dynamic Characterization of Cancer-Associated Variants of DNA Polymerase Beta

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有20,000个碱基对受损。如果不进行修复，这些受损的位点可能在复制时导致不匹配的诱导突变，最终导致癌症。幸运的是，细胞拥有非常有效的过程来检测、移除和替换这些病变。在这一过程中至关重要的酶之一是DNA聚合酶（Pol β），它负责将适当的核苷酸插入先前切除受损碱基对产生的间隙中，以确保保真度。因此，与“正常”或野生型（WT） Pol β相比，保真度或活性降低的Pol β突变体会导致癌症。尽管许多这样的突变体已经具有生物化学特征，但WT Pol β的微小结构变化如何显著改变Pol β的功能仍不清楚。在DNA修复过程中，Pol β发生了一系列大规模的、可逆的构象变化。传统上认为这些变化只发生在有底物存在的情况下。然而，最近，我们和其他人发现，在催化途径的所有阶段，即使在没有底物的情况下，WT Pol β也会自行经历快速、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异中发生了改变。该项目的目标是通过使用溶液状态弛豫色散核磁共振技术表征诱变Pol β变异的动力学来测试这一假设，并将这些研究结果与WT Pol β的动力学和先前的研究结果进行比较，这些研究已经表征了每种变异诱导的各种突变。

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.