MECHANISM AND REGULATION OF NONHOMOLOGOUS DNA ENDJOINING

DNA非同源连接的机制和调控

• 批准号: 6596588
• 负责人: MICHAEL R LIEBER
• 金额: $ 32.54万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6596588
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; active sites; aging; cell cycle; enzyme activity; gene mutation; gene targeting; genetically modified animals; inositol phosphates; laboratory mouse; neoplasm /cancer genetics; phosphorylation; protein purification; site directed mutagenesis; surface plasmon resonance; western blottings

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.

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，并且可能对衰老很重要。 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 DNA 链上；在后一种情况下，会导致双链 DNA 断裂。 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)。 该提案致力于在生化和遗传水平上了解 NHEJ 通路。 NHEJ途径是修复细胞周期G0、G1和早期S期双链DNA断裂的主要途径；因此，了解 NHEJ 具有广泛的医学意义。 该提案的主要工作是了解 NHEJ 通路中关键步骤的生物化学，并用生化纯化的蛋白质重建该通路。 在具体目标 1 中，我们测试了与 NHEJ 最早步骤相关的四个假设。 首先，我们检查 Ku 是否将 Artemis:DNA-PKcs 复合物招募到 DNA 末端。 在第二部分中，我们研究了磷酸肌醇可能调节 NHEJ 的点。 在第三部分中，我们研究了蛋白质磷酸化如何影响最初的蛋白质复合物，这些蛋白质被认为与断裂的 DNA 末端结合。 第四，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。 在具体目标 2 中，我们研究了两个断裂的 DNA 末端如何在物理上接近。 具体来说，我们测试这是否可以通过 Ku:Artemis:DNAPKcs 复合体来实现。 在具体目标 3 中，我们描述了使用纯化蛋白重建整个人类 NHEJ 通路的努力。 作为这一目标的一部分，我们还测试了 DNA 聚合酶的作用，DNA 聚合酶是高等真核生物中 NHEJ 尚未明确确定的一种酶活性。 具体目标 4 旨在确定 NHEJ 途径中主要核酸酶 Artemis 的活性位点。 这一目标还旨在了解 Artemis 和 DNA-PKcs 之间的相互作用区域。 总体而言，该提案代表了一项重大的共同努力，旨在加深和完善我们对修复双链 DNA 断裂的主要途径的理解。 生化定义的 NHEJ 系统的长期医学益处包括能够测试小分子药物抑制剂在癌症治疗中的作用。