EPIGENETIC CONTROL OF NHEJ DNA REPAIR

描述（由申请人提供）： 转座酶活性被认为在人类中已经灭绝，因为DNA移动在高等生物中可能是有害的，导致基因组不稳定性和可能的恶性肿瘤。然而，我们分离出一种称为Metnase的人转座酶蛋白，其具有作为内切核酸酶的转座酶结构域和SET组蛋白甲基化酶结构域。转座酶结构域对超螺旋DNA具有优先的核酸内切酶活性，并且SET结构域能够在与开放染色质相关的赖氨酸36处甲基化组蛋白3。Metnase增强对放射疗法的抗性，并通过非同源末端连接途径（NHEJ）改善DNA双链断裂（DSB）的修复。SET和转座酶结构域都是NHEJ修复活性所需的。 发现Metnase与NBS 1（早期NHEJ修复途径组分）、DNA连接酶IV（NHEJ途径的最终组分）和Pso 4（未表征的DSB修复组分）相互作用。我们发现Metnase降低了修复的DSB连接位点长缺失的发生率。Metnase在S495处被磷酸化，这对其NHEJ修复活性是必需的。我们表明，当不同染色体上同时存在DSB时，Metnase降低了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还介导对Topo II毒物的抗性，这会导致癌细胞系中的DSB。因此，Metnase是NHEJ修复途径的新组分，并将组蛋白修饰与DSB修复联系起来。 Metnase改善DNA修复的机制尚不清楚，但其SET组蛋白甲基化酶结构域对其NHEJ活性至关重要。最近的研究表明，染色质的修饰，包括组蛋白甲基化，可能是DSB修复的重要组成部分。然而，尽管其潜在的重要性，这种表观遗传染色质修饰和NHEJ DSB修复之间的联系还没有很好地定义。使用一种新的ChIP测定来分析与单个定义的DSB相关的蛋白质，我们发现Metnase定位于该DSB，并且在那里二甲基化H3 K36。我们还发现证据表明，二甲基化H3 K36可能会招募早期NHEJ组件，如ATM和MRN复合物，DSB。基于这些数据，我们假设Metnase在NHEJ DSB修复的表观遗传调控中起重要作用。这一假设将在四个目标中进行探索，这些目标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化酶活性至关重要？2)Metnase在DSB位点周围的组蛋白改变是什么？3)Metnase的组蛋白甲基化增强DSB修复的机制是什么？4)Metnase的组蛋白甲基化酶活性能否用于临床？ 公共卫生相关性： 我们已经分离出一种名为Metnase的新型蛋白质，它可以帮助修复断裂的染色体，从而防止可能导致癌症的突变的形成。Metnase可以通过用一种编码标记断裂的染色体来实现这一点，这种编码可以将修复装置招募到断裂处，从而增强修复。然而，癌细胞可以破坏Metnase，并利用它来抵抗放射治疗的作用。

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.