EPIGENETIC CONTROL OF NHEJ DNA REPAIR

NHEJ DNA 修复的表观遗传控制

• 批准号: 8007448
• 负责人: Robert A Hromas
• 金额: $ 20.37万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007448
• 关键词: Adriamycin PFS; Affect; Biological Assay; Breast Cancer Cell; CHES1 gene; Cancer Etiology; Cancer cell line; Chromatin; Chromosomal Breaks; Chromosomal translocation; Chromosomes; Clinical; Code; Complex; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA ligase IV; Data; Double Strand Break Repair; Epigenetic Process; Event; Genomic Instability; Histones; Human; Incidence; Ionizing radiation; Link; Lysine; Malignant Neoplasms; Mediating; Methylation; Methyltransferase; Molecular; Movement; Mutate; Mutation; NBS1 gene; Nonhomologous DNA End Joining; Organism; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Play; Poison; Process; Proteins; Radiation therapy; Recruitment Activity; Regulation; Resistance; Role; SET Domain; Screening procedure; Series; Site; Structure; Structure-Activity Relationship; Superhelical DNA; Testing; Time; Topoisomerase II; Translating; Transposase; Yeasts; base; cancer cell; chromatin immunoprecipitation; chromatin modification; clinically relevant; drug testing; endonuclease; histone methyltransferase; histone modification; improved; in vitro Assay; inhibitor/antagonist; insight; mutant; novel; nuclease; prevent; public health relevance; radiation resistance; repaired; virtual

DESCRIPTION (provided by applicant): Transposase activity was thought to be extinct in humans because DNA movement can be deleterious in higher organisms, resulting in genomic instability and perhaps malignancy. However, we isolated a human transposase protein termed Metnase that had both a Transposase domain that functions as an endonuclease and a SET histone methylase domain. The Transposase domain has preferential endonuclease activity for supercoiled DNA, and the SET domain was able to methylate histone 3 at lysine 36, associated with open chromatin. Metnase enhances resistance to radiation therapy, and improves repair of DNA double strand breaks (DSBs) via the non-homologous end-joining pathway (NHEJ). Both the SET and Transposase domains were required for the NHEJ repair activity. Metnase was found to interact with the NBS1, an early NHEJ repair pathway component, DNA Ligase IV, a final component of the NHEJ pathway, and Pso4, an uncharacterized DSB repair component. We found that Metnase decreases the incidence of long deletions at the repaired DSB junction site. Metnase is phosphorylated at S495, and this is essential for its NHEJ repair activity. We show that Metnase decreases the rate of inter-chromosomal translocation when there are simultaneous DSBs on distinct chromosomes, consistent with its NHEJ repair activity. We also found that Metnase also mediates resistance to Topo II poisons, which cause DSBs, in cancer cell lines. Thus, Metnase is a novel component of the NHEJ repair pathway, and links histone modification to DSB repair. The mechanism by which Metnase improves DNA repair is unknown, but its SET histone methylase domain is essential to its NHEJ activity. Recent studies in yeast indicate that the modification of chromatin, including histone methylation, may be an important part of DSB repair. However, despite its potential importance, the connection between such epigenetic chromatin modifications and NHEJ DSB repair is not well defined. Using a novel ChIP assay to analyze proteins associated with a single defined DSB, we found that Metnase localized to that DSB, and dimethylated H3K36 there. We also found evidence that dimethylated H3K36 may recruit early NHEJ components, such as ATM and the MRN complex, to the DSB. Based on these data, we hypothesize that Metnase plays an important role in the epigenetic regulation of NHEJ DSB repair. This hypothesis will be explored in four aims that translate molecular mechanisms to clinical relevance- 1) What are the structures of Metnase that are essential for its histone methylase activity? 2) What are the histone alterations Metnase makes around a DSB sites? 3) What is the mechanism by which Metnase's histone methylation enhances DSB repair? 4) Can the histone methylase activity of Metnase be exploited clinically? PUBLIC HEALTH RELEVANCE: We have isolated a novel protein termed Metnase that helps broken chromosomes repair, and thereby prevent the formation of mutations that can cause cancer. Metnase may do this by marking the broken chromosome with a code that recruits the repair apparatus to the break, enhancing repair. The cancer cell, however, can subvert Metnase, and use it to resist the actions of radiation therapy.

描述（由申请人提供）： 人们认为转座酶活性在人类中已灭绝，因为DNA运动在较高的生物体中可能是有害的，从而导致基因组不稳定性和可能恶性肿瘤。但是，我们分离了一种称为metnase的人转座酶蛋白，该蛋白既具有转座酶结构域，该蛋白结构域起作用，该蛋白结构域起作用，可作为内核酸酶和固定的组蛋白甲基酶结构域。转座酶结构域具有超涂DNA的优先内核酸酶活性，并且SET结构域能够在赖氨酸36处甲基化组蛋白3，与开放染色质有关。 MetNase通过非同理端连接途径（NHEJ）提高了对放射疗法的抵抗力，并改善了DNA双链断裂（DSB）的修复。 NHEJ修复活性都需要集合和转座酶结构域。 MetNase与NBS1相互作用，NBS1是一种早期的NHEJ修复途径成分，DNA连接酶IV，NHEJ途径的最终组成部分，而PSO4是未表征的DSB修复分量。我们发现，Metnase降低了修复的DSB结位点长缺失的发生率。 MetNase在S495处被磷酸化，这对于其NHEJ修复活性至关重要。我们表明，当在不同的染色体上同时存在DSB时，Metnase会降低染色体间易位的速率，这与其NHEJ修复活性一致。我们还发现，Metnase还介导了对癌细胞系中引起DSB的Topo II毒物的抗性。因此，metnase是NHEJ修复途径的新成分，并将组蛋白的修饰与DSB修复联系起来。 Metnase改善DNA修复的机制尚不清楚，但其固定的组蛋白甲基酶结构域对于其NHEJ活性至关重要。酵母的最新研究表明，包括组蛋白甲基化在内的染色质的修饰可能是DSB修复的重要组成部分。然而，尽管具有潜在的重要性，但这种表观遗传染色质修饰与NHEJ DSB修复之间的联系并不是很好。使用新颖的芯片测定法分析与单个定义的DSB相关的蛋白质，我们发现Metnase位于该DSB中，并在那里二甲基化的H3K36。我们还发现证据表明，二甲基化的H3K36可能会将早期的NHEJ组件（例​​如ATM和MRN复合物）募集到DSB。基于这些数据，我们假设Metnase在NHEJ DSB修复的表观遗传调节中起重要作用。将在四个目标中探讨该假设，将分子机制转化为临床相关性-1）Metnase的结构对其组蛋白甲基酶活性至关重要？ 2）Metnase在DSB站点周围产生了哪些组蛋白改变？ 3）Metnase的组蛋白甲基化可以增强DSB修复的机制？ 4）临床上可以利用metnase的组蛋白甲基化酶活性吗？ 公共卫生相关性： 我们已经分离出一种称为metnase的新型蛋白质，该蛋白有助于破裂的染色体修复，从而防止形成可能引起癌症的突变。 Metnase可以通过用代码标记损坏的染色体来做到这一点，该代码将修复设备募集到休息中，从而增强维修。然而，癌细胞可以颠覆metnase，并使用它来抵抗放射治疗的作用。