Mechanism and Regulation of Nonhomologous DNA End Joining

描述（由申请人提供）：双链DNA断裂（DSBs）每天在所有细胞中发生多次。同源重组（HR）和非同源DNA末端连接（NHEJ）是修复dsb的两种主要途径。然而，NHEJ是更普遍的途径，因为它不需要同源供体，而且NHEJ具有足够的灵活性，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的自然原因（电离辐射、自由基、酶功能障碍）产生具有不同化学和结构构型的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在其发挥作用的大多数位点（在人类和其他脊椎动物中）导致DNA序列改变。因此，NHEJ产生导致癌症的体细胞突变，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。虽然我们知道大多数参与NHEJ的蛋白质，并且从广义上知道它们的作用，但我们并不清楚它们是如何一起起作用的，它们的空间结构，或者它们的时间关系。有了更清晰的人类NHEJ的机制和结构图，我们将能够开发出可能用于治疗癌症的小分子抑制剂（作为化疗或辐射增敏剂）。本提案的目的1和2集中在NHEJ蛋白之间以及与DNA末端的结构和空间关系的问题上。目的3验证了NHEJ连接酶复合物对于在NHEJ过程中进行关键的端到端接触很重要的假设。目标4描述了一个更健壮的NHEJ重构的发展。在目标4中，该系统随后通过测试活性增强来识别任何剩余的NHEJ因子。目的5描述了组蛋白八聚体如何影响NHEJ的核酸酶、聚合酶和连接酶活性的分析。这些研究将显著提高我们对NHEJ如何参与对癌症病因至关重要的染色体易位等过程的理解，以及如何将NHEJ制成“可药物”用于治疗目的。

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.