Non-Homologous End Joining Repair in Humans

描述（由申请人提供）：人类基因组中散布着来自Hsmar 1转座子的转座因子的序列，但只有一个完整拷贝的Hsmar 1转座酶基因，称为Metnase（也称为SETMAR），存在于嵌合SET转座酶融合蛋白中。虽然Metnase保留了大部分转座酶活性，但它已在灵长类动物中进化为双链断裂（DSB）修复蛋白。Metnase位于染色体3 p26上，这是各种癌症中常见的异常区域，并且在大多数组织和细胞系中高度表达。在许多转化细胞系中发现了导致早期终止的Metnase突变，尽管这些突变的临床相关性尚未确定。我们的长期目标是了解人类中具有转座酶活性的蛋白质如何促进DSB修复和染色体去连锁，以及SET结构域可能发挥什么作用。鉴于Metnase需要SET和转座酶结构域用于其在DSB修复中的功能，我们假设新功能的获得可能是由转座酶和SET结构域之间的嵌合融合引起的。在这项研究中，我们提出了三个具体的目标，以阐明这种人类SET转座酶蛋白在DSB修复和染色体脱连锁的机制。目的1：确定Metnase定位于DSB位点的机制。我们将鉴定在IR处理后对DSB位点定位至关重要的Metnase的SET结构域内的区域。我们还将研究Metnase与Pso 4（在DSB位点的Metnase定位中起关键作用的Metnase结合伴侣）的相互作用是否对DSB位点的Metnase定位至关重要。最后，我们将研究Metnase是否直接与Ku 70/80复合物相互作用。如果是这样的话，将产生与Ku复合物相互作用有缺陷的突变体Metnase，并且我们将确定该突变体在DSB位点的定位方面与wt-Metnase有何不同。目的2：探讨Metnase的生化活性在DNA末端连接和染色体脱连锁中的作用。Metnase不仅具有结构特异性内切酶和HLMT活性，还与Lig 4、Pso 4和Topo II 1相互作用，在NHEJ修复和/或染色体脱连锁中发挥作用。我们将研究Metnse的生化活动是如何参与DNA末端连接和染色体去连锁的。首先，我们将取代从Metnase转座酶结构域的晶体结构中鉴定的催化位点内的关键氨基酸，并检查突变体的DNA切割、DNA末端加工和末端连接活性。其次，我们将研究Metnase与Lig 4的相互作用如何影响Lig 4-XRCC 4向DSB位点的募集和DNA末端连接。第三，我们将研究Metnase结合伴侣（Pso 4）及其与Metnase的相互作用如何影响DNA末端连接。第四，我们将检查缺乏HLMT和/或自动甲基化活性的Metnase突变体是否支持DSB修复的刺激。最后，将检查缺乏其生物化学活性的Metnase突变体对染色体去连锁活性的促进作用。 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，它有助于修复人类辐射诱导的染色体断裂和染色体脱连锁。了解Metnase及其结合伴侣有助于修复染色体断裂和去连锁的分子机制不仅有助于了解人类基因组的稳定维持，而且有助于开发抑制癌细胞中染色体DNA修复和去连锁的药物。

DESCRIPTION (provided by applicant): The human genome is littered with sequences derived from transposable elements from the Hsmar1 transposon, but there is only one intact copy of the Hsmar1 transposase gene termed Metnase (also known as SETMAR) that exists within a chimeric SET-transposase fusion protein. Although Metnase retains most of the transposase activities, it has evolved as a double-strand break (DSB) repair protein in anthropoid primates. Metnase is localized on chromosome 3p26, a region of frequent abnormalities in various cancers and is highly expressed in most tissues and cell lines. Mutations in Metnase that cause early termination were found in many transformed cell lines, although clinical relevance of these mutations has not been established. Our long-term goal is to understand how a protein with transposase activity in humans promotes DSB repair and chromosome decatenation, and what role the SET domain may play. Given that Metnase requires both the SET and transposase domains for its function(s) in DSB repair, we hypothesize that the acquisition of new functions may have resulted from a chimeric fusion between transposase and the SET domains. In this study we proposed three specific aims to elucidate the mechanism of this human SET- transposase protein in DSB repair and chromosome decatenation. Aim 1: Determine the mechanism by which Metnase is localized to DSB sites. We will identify the region within the SET domain of Metnase crucial for localization to DSB sites following IR treatment. We will also investigate whether the interaction of Metnase with Pso4, a Metnase binding partner that plays a crucial role in Metnase localization at DSB sites, is crucial for Metnase localization at DSB sites. Finally, we will examine whether Metnase directly interacts with the Ku70/80 complex. If so, a mutant Metnase defective in interaction with Ku complex will be generated, and we will determine how this mutant differs from wt-Metnase in their localization at DSB sites. Aim 2: Determine the role(s) of Metnase's biochemical activities in DNA end joining and chromosome decatenation. Metnase not only possesses a structure-specific endonuclease and HLMT activities, but also interacts with Lig4, Pso4, and Topo II1, all of which could play role(s) in NHEJ repair and/or chromosome decatenation. We will examine how Metnse's biochemical activities are involved in DNA end joining and chromosome decatenation. First, we will substitute key amino acids within the catalytic site identified from the crystal structure of Metnase transposase domain and examine the mutants for DNA cleavage, DNA end processing, and end joining activities. Secondly, we will examine how Metnase's interaction with Lig4 affects recruitment of Lig4-XRCC4 to DSB sites and DNA end joining. Thirdly, we will examine how Metnase binding partner (Pso4) and its interaction with Metnase influence DNA end joining. Fourth, we will examine whether Metnase mutant(s) lacking HLMT and/or auto-methylation activity support stimulation of DSB repair. Finally, Metnase mutants lacking its biochemical activities will be examined for promotion of chromosome decatenation activity. PUBLIC HEALTH RELEVANCE: We have isolated a novel protein termed Metnase that helps repair of radiation-induced chromosomes breaks and chromosome decatenation in humans. Understanding the molecular mechanism by which Metnase and its binding partners contribute to repair of chromosomal breaks and decatenation could not only help understand stable maintenance of human genome but also be useful in developing a drug that inhibits chromosomal DNA repair and decatenation in cancer cells.