Mechanism and Regulation of Nonhomologous DNA End Joining

描述(申请人提供)：双链DNA断裂(DSB)在所有细胞中每天发生多次。同源重组(HR)和非同源DNA末端连接(NHEJ)是修复DSB的两条主要途径。然而，NHEJ是更一般的途径，因为它不需要同源供体，并且NHEJ足够灵活，它可以连接几乎任何一对不相容的DNA末端。NHEJ的灵活性是必不可少的，因为DSB(电离辐射、自由基、酶故障)的自然原因会产生具有不同化学和结构配置的DNA末端。因此，这种灵活性非常适合这项任务，但我们为这种灵活性付出的代价是，NHEJ会在它起作用的大多数部位引起DNA序列变化(在人类和其他脊椎动物中)。因此，NHEJ产生的体细胞突变会导致癌症，并可能导致衰老。NHEJ的灵活性代表了最复杂的蛋白质之一：DNA相互作用途径。尽管我们知道大多数参与NHEJ的蛋白质，并从广义上了解它们的功能，但我们并不清楚它们如何共同发挥作用，它们的空间构型，或时间关系。随着对人类NHEJ机制和结构的更清楚的了解，我们将能够开发出可能对治疗癌症有用的小分子抑制剂(作为化疗或辐射增敏剂)。这项提案的目标1和2正好集中在NHEJ蛋白和DNA末端之间的结构和空间关系问题上。目的3验证了NHEJ连接酶复合体在NHEJ期间进行关键的端到端接触的假设。目标4描述了更强大的NHEJ重建的发展。在目标4中，然后使用该系统通过活性增强测试来识别任何剩余的NHEJ因子。目的5分析组蛋白八聚体如何影响NHEJ的核酸酶、聚合酶和连接酶活性。这些研究将显著提高我们对NHEJ如何参与染色体易位等过程的理解，这些过程在癌症病因学中具有关键意义，以及如何使NHEJ可用于治疗目的。 与公共健康相关：这个项目将澄清我们对修复DNA损伤的关键途径之一的见解。这些见解将使我们了解一些导致癌症和衰老的DNA损伤是如何发生的。通过更深入地了解这一关键的DNA修复途径，可以增加一些化疗和治疗性放射工作的好处。

DESCRIPTION (provided by applicant): Double-strand DNA breaks (DSBs) occur in all cells multiple times per day. Homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ) are the two primary pathways for repairing DSBs. However, NHEJ is the more general pathway because it does not require a homologous donor, and NHEJ is sufficiently flexible that it can join almost any pair of incompatible DNA ends. The flexibility of NHEJ is essential because natural causes of DSBs (ionizing radiation, free radicals, enzyme malfunction) generate DNA ends with diverse chemical and structural configurations. Hence, that flexibility is well-suited for the task, but the price that we pay for that flexibility is that NHEJ causes DNA sequence changes at most sites where it functions (in humans and other vertebrates). Hence, NHEJ generates somatic cell mutations that cause cancer and likely contribute to aging. The flexibility of NHEJ represents one of the most sophisticated protein:DNA interaction pathways. Though we know most of the proteins that participate in NHEJ and know, in broad terms, what they do, we do not have a clear picture of how they function together, their spatial configuration, or the temporal relationships. With a clearer mechanistic and structural picture of human NHEJ, we will be in a position to develop small molecule inhibitors that may be useful for treating cancer (as chemotherapy or as a radiation sensitizer). Aims 1 and 2 of this proposal focus precisely on the issues of structural and spatial relationships among the NHEJ proteins and with the DNA ends. Aim 3 tests the hypothesis that the NHEJ ligase complex is important for making key end-to-end contacts during NHEJ. Aim 4 describes development of a more robust NHEJ reconstitution. In Aim 4, that system is then used to identify any remaining NHEJ factors by testing for enhancement of activity. Aim 5 describes analysis of how histone octamers influence the nuclease, polymerase, and ligase activities of NHEJ. These studies will markedly improve our understanding of how NHEJ participates in processes such as chromosomal translocations that are of key importance in cancer etiology and how NHEJ might be made 'druggable' for therapeutic purposes. PUBLIC HEALTH RELEVANCE: This project would clarify our insights into one of the key pathways for the repair of DNA damage. These insights will allow us to understand how some of the DNA damage that causes cancer and aging occurs. The benefits of some chemotherapy and therapeutic radiation work can be increased by this deeper level of understanding of this key DNA repair pathway.