Regulation of Single Strand Annealing Repair of Mammalian Chromosomal Breaks

 说明(申请人提供)：电离辐射(IR)是一种致裂剂，因为IR暴露会导致DNA双链断裂(DSB)。在接触断裂剂后，未能恢复原始DNA序列会导致遗传损失，这可能是这些制剂的细胞毒性的原因之一。Clastogen诱导的遗传信息丢失可能是DSB修复的结果，这种修复是非恢复性的，就像单链退火(SSA)修复所发生的那样。也就是说，SSA是一种同源重组(HR)途径，它使用侧翼同源重复序列来连接DSB，从而导致缺失重排，并丢失重复之间的遗传信息。SSA有可能对哺乳动物基因组的完整性造成灾难性的影响，因为人类基因组中含有大量的重复元素，包括大约100万个Alu类元素。相反，另一种HR途径，同源定向修复(HDR)，相对具有恢复性，因为它使用精确的姐妹染色单体作为模板。我们的总体假设是，SSA可以防止染色体丢失，但调节这一途径有利于HDR而不是SSA，并限制由SSA引起的缺失重排的大小，对于裂解原抗性是重要的。我们已经确定了两个因素对SSA的调节很重要：四肽重复蛋白XAB2是SSA的媒介，而E3泛素连接酶RNF168抑制SSA的方式在HR媒介BRCA1缺失的细胞中被放大。为了了解SSA的调节，我们的具体目的是：目的1.确定XAB2的SSA介体功能。我们将检验这样的假设，即XAB2促进HR(HDR和SSA)的末端切除步骤，以及XAB2对HR重要的基序对于其与另一个HR介体的相互作用至关重要， PRP19.我们还将验证这样的假设，即缺乏SSA的亚型XAB2突变体可以保留介导恢复性HDR途径的功能，从而也擅长促进碎裂原抗性。目的2.明确RNF168的SSA抑制功能。我们将检验这一假设，即RNF168与一种SSA抑制剂(H2AX)在相同的途径上发挥作用，但与另一种途径(Ku70，c-NHEJ途径)不同。此外，我们将检验RNF168 C末端含有限制其抗SSA活性的负调控结构域的假设。目的3.研究DSB和重复序列之间的距离如何影响SSA的频率和调节。为此，我们将开发一种新的报告程序，用于重复介导的删除重排，其中还将包括重复序列分歧的变异。这与理解遗传损失有关，因为DSB/重复距离定义了由SSA引起的缺失的大小。这个项目意义重大，因为它将揭示影响碎裂原暴露造成的遗传损失的机制。这项工作的创新之处在于对XAB2和RNF168在基因组维护过程中功能的新见解，以及一种独特的报告系统来检查重复介导的删除重排。

 DESCRIPTION (provided by applicant): Ionizing radiation (IR) is a clastogenic agent, in that IR exposure causes DNA double-strand breaks (DSBs). Failure to restore the original DNA sequence following clastogen exposure causes genetic loss, which likely contributes to the cellular toxicity of these agents. Clastogen-induced loss of genetic information can be a consequence of DSB repair that is non-restorative, as occurs with Single Strand Annealing (SSA) repair. Namely, SSA is a type of homologous recombination (HR) pathway that uses flanking homologous repeat sequences to bridge the DSB, which causes a deletion rearrangement, with loss of the genetic information between the repeats. SSA has the potential to be catastrophic to the integrity of mammalian genomes, given the high level of repetitive elements, including the approximately one million Alu-type elements in the human genome. In contrast, another HR pathway, Homology-Directed Repair (HDR), is relatively restorative, since it uses the precise sister chromatid as a template. Our overall hypothesis is that SSA protects against chromosome loss, but regulation of this pathway to favor HDR vs. SSA, and limit the size of deletion rearrangements caused by SSA, are important for clastogen resistance. We have identified two factors as being important for the regulation of SSA: the tetratricopeptide repeat protein XAB2 is a mediator of SSA, whereas the E3 ubiquitin ligase RNF168 inhibits SSA in a manner that is magnified in cells depleted of the HR mediator BRCA1. To understand the regulation of SSA, our specific aims are: Aim 1. To define the SSA mediator function of XAB2. We will test the hypotheses that XAB2 promotes the end resection step of HR (HDR and SSA), and that motifs of XAB2 important for HR are critical for its interaction with another HR mediator, PRP19. We will also test the hypothesis that hypomorphic XAB2 mutants deficient for SSA can retain function to mediate the restorative HDR pathway, and hence also be proficient at promoting clastogen resistance. Aim 2. To define the SSA inhibition function of RNF168. We will test the hypothesis that RNF168 functions in the same pathway as one SSA inhibitor (H2AX), but is distinct from another (Ku70, c-NHEJ pathway). Furthermore, we will test the hypothesis that the RNF168 C-terminus contains a negative regulatory domain that limits its anti-SSA activity. Aim 3. To examine how the distance between DSBs and repeat sequences affects the frequency and regulation of SSA. For this, we will develop a novel reporter for repeat-mediated deletion rearrangements, which will also include variations in repeat sequence divergence. This is relevant to understanding genetic loss, since the DSB/repeat distance defines the size of the deletion caused by SSA. This project is significant, as it will uncover mechanisms that influence genetic loss caused by clastogen exposure. The innovation in this work lies in the novel insight into XAB2 and RNF168 function during genome maintenance, and a unique reporter system to examine repeat-mediated deletion rearrangements.