MECHANISM AND REGULATION OF NONHOMOLOGOUS DNA ENDJOINING

描述(由申请人提供)：获得性基因突变是所有形式癌症的基础，很可能在衰老过程中起重要作用。DNA损伤是突变的主要原因，可能发生在一条DNA链上，也可能发生在两条链上；在后一种情况下，会导致双链DNA断裂。在多细胞真核生物中，双链断裂修复的两种形式是同源重组(HR)和非同源DNA末端连接(NHEJ)。这项建议致力于在生化和遗传水平上理解NHEJ途径。NHEJ途径是细胞周期G0期、G1期和S早期修复双链DNA断裂的主要途径，因此，对NHEJ的理解具有广泛的医学意义。这项建议的主要工作是了解NHEJ途径关键步骤的生物化学，并用生化纯化的蛋白质重建该途径。在具体目标1中，我们检验了与NHEJ中最早的步骤相关的四个假设。首先，我们考察Ku是否将Artemis：DNA-PKcs复合体招募到DNA末端。在第二部分中，我们研究了肌醇磷酸可能调节NHEJ的点。在第三个实验中，我们研究了蛋白质磷酸化如何影响蛋白质的初始复合体，这些蛋白质被认为是结合在DNA断裂的末端。在第四个实验中，我们使用小鼠模型来测试NHEJ通路早期阶段蛋白质之间的干扰。在特定的目标2中，我们研究了两个断裂的DNA末端是如何进入物理上的接近的。具体地说，我们测试Ku：Artemis：DNAPKcs复合体是否可以实现这一点。在具体目标3中，我们描述了使用纯化的蛋白重建整个人类NHEJ途径的努力。作为这一目标的一部分，我们还测试了DNA聚合酶的作用，这是NHEJ在高等真核生物中尚未确定的一种酶活性。具体目标4旨在定义NHEJ途径中主要核酸酶Artemis的活性部位。这一目的也是为了了解Artemis和DNA-PKcs之间的相互作用区域。总体而言，这项建议代表了一项重大的协调努力，以加深和完善我们对修复双链DNA断裂这一主要途径的理解。生物化学定义的NHEJ系统的长期医疗益处包括测试小分子药物抑制剂在癌症治疗中的作用的能力。

DESCRIPTION (provided by applicant): Acquired genetic mutations underlie all forms of cancer and are likely to be important in aging. DNA damage, a major cause of mutations, can occur on one DNA strand or both strands; in the latter case, a double-strand DNA break results. The two forms of double-strand break repair in multicellular eukaryotes are homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). This proposal is devoted to understanding the NHEJ pathway at the biochemical and genetic levels. The NHEJ pathway is the major pathway for repairing double-strand DNA breaks during G0, G1, and early S phases of the cell cycle; therefore, the understanding of NHEJ is of broad medical importance. The major effort of this proposal is to understand the biochemistry of key steps in the NHEJ pathway and to reconstitute the pathway with biochemically purified proteins. In Specific Aim 1, we test four hypotheses related to the earliest steps in NHEJ. In the first, we examine whether Ku recruits the Artemis:DNA-PKcs complex to a DNA end. In the second, we examine the point at which inositol phosphates might regulate NHEJ. In the third, we examine how protein phosphorylation affects the initial complex of proteins that are thought to bind at a broken DNA end. In the fourth, we use a murine mouse model to test for interference between proteins in the early phase of the NHEJ pathway. In Specific Aim 2, we examine how two broken DNA ends are brought into physicial proximity. Specifically, we test whether this can be achieved by the Ku:Artemis:DNAPKcs complex. In Specific Aim 3, we describe efforts to reconstitute the entire human NHEJ pathway using purified proteins. As part of this aim, we also test for roles of DNA polymerases, which are the one type of enzymatic activity yet to be definitively determined for NHEJ in higher eukaryotes. Specific Aim 4 is directed at defining the active site for the major nuclease in the NHEJ pathway, Artemis. This aim is also directed at understanding the region of interaction between Artemis and DNA-PKcs. Overall, this proposal represents a major concerted effort to deepen and complete our understanding of this primary pathway of repairing double-strand DNA breaks. The long-term medical benefit of a biochemically-defined NHEJ system includes the ability to test small molecule drug inhibitors for roles in cancer therapy.