Regulation of Single Strand Annealing Repair of Mammalian Chromosomal Breaks

哺乳动物染色体断裂单链退火修复的调控

• 批准号: 9236171
• 负责人: Jeremy Michael Stark
• 金额: $ 38.89万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9236171
• 关键词: Affect; Alu Elements; BRCA1 gene; Biological Assay; Cell Line; Cells; Chromosomal Breaks; Chromosomes; DNA; DNA Double Strand Break; DNA Sequence; Disease; Double Effect; Double Strand Break Repair; Elements; Event; Excision; Failure; Frequencies; G22P1 gene; Genetic; Genome; Goals; Human Genome; Ionizing radiation; Maintenance; Mediating; Mediator of activation protein; Mission; Mus; N-terminal; Nonhomologous DNA End Joining; Pathway interactions; Patient-Focused Outcomes; Proteins; Public Health; Radiation exposure; Regulation; Regulatory Element; Repetitive Sequence; Reporter; Research; Resistance; Role; Sister Chromatid; System; Testing; Therapeutic; Toxic effect; Tumor Suppressor Proteins; United States National Institutes of Health; Variant; Work; base; cancer therapy; chromosome loss; clastogen; embryonic stem cell; genetic information; homologous recombination; improved; inhibitor/antagonist; innovation; insight; mammalian genome; mutant; novel; novel therapeutic intervention; nuclease; public health relevance; repaired; response; tumor; ubiquitin-protein ligase

 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.

 描述（由申请人提供）：电离辐射（IR）是一种致染色体断裂剂，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序列会导致遗传丢失，这可能导致这些药剂的细胞毒性。断裂素诱导的遗传信息丢失可能是DSB修复的结果，其是非恢复性的，如单链退火（SSA）修复所发生的。也就是说，SSA是一种同源重组（HR）途径，其使用侧翼同源重复序列来桥接DSB，这导致缺失重排，其中重复之间的遗传信息丢失。SSA有可能对哺乳动物基因组的完整性造成灾难性的影响，因为人类基因组中存在高水平的重复元件，包括大约100万个重复型元件。相比之下，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防止染色体丢失，但该途径的调节有利于HDR与SSA，并限制SSA引起的缺失重排的大小，对于染色体断裂剂抗性是重要的。我们已经确定了两个因素是重要的SSA的调节：的tetratricopeptide重复蛋白XAB 2是介质的SSA，而E3泛素连接酶RNF 168抑制SSA的方式是放大的细胞中耗尽的HR介质BRCA 1。为了理解SSA的监管，我们的具体目标是：目标1。定义XAB 2的SSA中介功能。我们将检验以下假设：XAB 2促进HR的末端切除步骤（HDR和SSA），以及XAB 2的基序对HR重要，对于其与另一种HR介导物的相互作用至关重要， PRP19。我们还将测试以下假设：SSA缺陷的亚纯型XAB 2突变体可以保留介导恢复性HDR途径的功能，因此也精通促进断裂素抗性。目标二。明确RNF 168的SSA抑制功能。我们将测试RNF 168在与一种SSA抑制剂（H2 AX）相同的途径中起作用，但与另一种（Ku 70，c-NHEJ途径）不同的假设。此外，我们将测试的假设，RNF 168的C-末端包含一个负调控域，限制其抗SSA活性。目标3.目的：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重复介导的缺失重排，这也将包括重复序列分歧的变化。这与理解遗传丢失有关，因为DSB/重复距离定义了SSA引起的缺失的大小。该项目意义重大，因为它将揭示影响染色体断裂剂暴露引起的遗传损失的机制。这项工作的创新在于对XAB 2和RNF 168在基因组维护过程中的功能的新见解，以及一种独特的报告系统来检查重复介导的缺失重排。