Non-Homologous End Joining Repair in Humans

描述（由申请人提供）：人类基因组中充斥着来自Hsmar1转座子的转座元件的序列，但是只有一个Hsmar1转座酶基因的完整拷贝，称为Metnase（也称为SETMAR），存在于嵌合的set -转座酶融合蛋白中。虽然Metnase保留了大部分转座酶的活性，但它已在类人猿中进化为双链断裂（DSB）修复蛋白。甲基化酶位于染色体3p26上，这是各种癌症中常见的异常区域，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甲基化酶突变，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在人类中具有转座酶活性的蛋白质如何促进DSB修复和染色体十烷化，以及SET结构域可能发挥的作用。鉴于Metnase在DSB修复中的功能需要SET和转座酶结构域，我们假设新功能的获得可能是由于转座酶和SET结构域之间的嵌合融合。在这项研究中，我们提出了三个具体的目的来阐明这种人类SET-转座酶蛋白在DSB修复和染色体十烷化中的机制。目的1：确定Metnase定位到DSB位点的机制。我们将在Metnase的SET域中确定对IR治疗后定位到DSB位点至关重要的区域。我们还将研究Metnase与Pso4的相互作用是否对Metnase在DSB位点的定位至关重要，Pso4是Metnase在DSB位点的定位至关重要的结合伙伴。最后，我们将研究Metnase是否直接与Ku70/80复合物相互作用。如果是这样，将产生一个与Ku复合物相互作用有缺陷的突变体Metnase，我们将确定该突变体与wt-Metnase在DSB位点的定位有何不同。目的2：确定甲基化酶的生化活性在DNA末端连接和染色体十烷化中的作用。Metnase不仅具有结构特异性内切酶和HLMT活性，而且还与Lig4、Pso4和Topo II1相互作用，所有这些都可能在NHEJ修复和/或染色体十烷化中发挥作用。我们将研究Metnse的生化活动如何参与DNA末端连接和染色体十烷化。首先，我们将替换从Metnase转座酶结构域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Lig4的相互作用如何影响Lig4- xrcc4在DSB位点的招募和DNA末端连接。第三，我们将研究Metnase结合伙伴（Pso4）及其与Metnase的相互作用如何影响DNA末端连接。第四，我们将研究缺乏HLMT和/或自甲基化活性的甲基化酶突变体是否支持刺激DSB修复。最后，缺乏生化活性的甲基化酶突变体将被检测以促进染色体十烷化活性。

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.