Mechanism of radial chromosome formation in human premature aging syndrome cells

人类早衰综合征细胞放射状染色体形成机制

基本信息

批准号：
10592123
负责人：
Anja-Katrin Bielinsky
金额：
--
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-01 至 2022-12-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10592123
关键词：
Aging Amaze Appearance Applications Grants Belief Bioinformatics Biological Models Bloom Syndrome Bone marrow failure Cell Aging Cell Line Cell physiology Cells Centromere Cessation of life Chromosomal translocation Chromosomes Cisplatin Conscious Cross-Linking Reagents Cytogenetics DNA Crosslinking DNA Crosslinking Agent DNA Damage DNA Double Strand Break DNA Repair DNA crosslink DNA ligase III DNA-Directed DNA Polymerase Data Diagnostic Diagnostic tests Double Strand Break Repair Exposure to Fanconi Anemia pathway Fanconi&apos s Anemia Frequencies Genes Genetic Recombination Genomic DNA Goals Grant Human Human Cell Line Individual Knowledge Lasers Literature Mediating Metaphase Microdissection Mission Mitomycin C Modeling Molecular Molecular Profiling Nonhomologous DNA End Joining Normal Cell Outcome Pathology Pathway interactions Patients Phenotype Polymerase Premature aging syndrome Process Production Protocols documentation Radial Radiation Regulation Reporting Research Personnel Role System Testing Therapeutic United States National Institutes of Health bioinformatics pipeline cancer predisposition chromosome fusion clinically relevant crosslink experimental study gene synthesis helicase insight nanopore new therapeutic target novel repaired response telomere ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Radial chromosomes (hereafter “radials”) have been part of the scientific consciousness for more than 50 years. Radials are operationally defined as fusions involving two or more nonhomologous chromosomes and as having more than one centromere. In normal cells, radials are quite rare, and they almost never form spontaneously. Radials have an important clinical relevance, however, since their appearance is a diagnostic hallmark of the bone marrow failure, cancer predisposition and premature aging syndrome, Fanconi anemia (FA). Interestingly though, even in FA cells, radial chromosomes do not appear spontaneously with high frequency but usually need to be induced by exposure to clinically-relevant DNA crosslinking reagents, such as mitomycin C (MMC) and cisplatin. Despite their diagnostic use for many decades, radial formation is still poorly understood, and the literature is filled with conflicting reports about what is required mechanistically for their production. In our first Specific Aim, we describe the construction of a human cell line that is defective for the radiation sensitive 18 (RAD18) gene. RAD18 encodes an E3 ubiquitin ligase and is required for translesion synthesis (TLS), an error-prone DNA damage tolerance pathway, which is a subpathway in the FA-mediated DNA crosslink repair process. Consistent with this role, RAD18-null human cells generated high levels of MMC- induced radials. Unexpectedly, the absence of DNA ligase III (LIGIII) and DNA polymerase theta (POLQ; both key components of alternative non-homologous end joining; A-EJ)) dramatically reduced RAD18-dependent, MMC-induced radial formation. Thus, the radials generated by the absence of RAD18 appear to require the A- EJ pathway. We propose to confirm these observations and extend the generality of these studies by determining the requirement for A-EJ in MMC-induced radial formation in Bloom syndrome (BLM) (another cancer predisposition and premature aging syndrome) and FA-defective cells. Amazingly, there is not a single report in the literature regarding the molecular makeup of a radial chromosome fusion junction. This is unfortunate, since an analysis of fusion junctions for a wide variety of other types of translocations (e.g., canonical reciprocal chromosomal translocations and telomere fusions) has provide a wealth of data regarding the molecular signatures of such junctions as well as insight into the repair mechanisms responsible for their formation. In our Specific Aim 2, we propose to rectify this egregious omission. Specifically, we describe a protocol (Radial-Seq) for isolating, sequencing and bioinformatically analyzing radial chromosome fusions. We expect this analysis to confirm the formation of such junctions by A-EJ and to greatly extend our understanding of the molecular makeup of a radial fusion. In toto, these experiments should mechanistically define the requirements for radial formation which should benefit basic researchers; in addition, the assignment of A-EJ for their formation may help clinicians therapeutically hinder their formation.

项目概要放射状染色体（以下简称“放射状”）50 多年来一直是科学意识的一部分径向在操作上被定义为两个或多个非同源染色体的融合。由于在正常细胞中具有多个着丝粒，放射状丝非常罕见，并且它们几乎从不形成。然而，桡动脉具有重要的临床意义，因为它们的外观具有诊断意义。骨髓衰竭、癌症易感性和早衰综合征、范可尼贫血的标志（FA）表明，即使在 FA 细胞中，放射状染色体也不会在高浓度下自发出现。频率，但通常需要通过接触临床相关的 DNA 交联试剂来诱导，例如丝裂霉素 C (MMC) 和顺铂尽管已用于诊断数十年，但放射状形成仍然很差。理解，并且文献中充满了关于他们机械地需要什么的相互矛盾的报告在我们的第一个具体目标中，我们描述了有缺陷的人类细胞系的构建。辐射敏感 18 (RAD18) 基因编码 E3 泛素连接酶，是跨损伤所必需的。合成（TLS），一种容易出错的 DNA 损伤耐受途径，是 FA 介导的 DNA 损伤耐受途径中的一个子途径 DNA 交联修复过程与此作用一致，RAD18 缺失的人类细胞产生了高水平的 MMC-。意外地，DNA 连接酶 III (LIGIII) 和 DNA 聚合酶 theta (POLQ；两者均缺失)。替代非同源末端连接的关键组成部分；A-EJ)) 显着减少了 RAD18 依赖性， MMC 诱导的径向形成因此，由于 RAD18 的缺失而产生的径向似乎需要 A-。我们建议通过以下方式证实这些观察结果并扩展这些研究的普遍性。确定布卢姆综合征 (BLM) 中 MMC 诱导的放射状形成对 A-EJ 的要求（另一个癌症易感性和早衰综合症）和 FA 缺陷细胞。令人惊讶的是，文献中没有一篇关于放射状分子组成的报道这是不幸的，因为对多种其他融合连接的分析。易位类型（例如，典型的相互染色体易位和端粒融合）提供了有关此类连接的分子特征以及对修复的深入了解的大量数据在我们的具体目标 2 中，我们建议纠正这一严重的遗漏。具体来说，我们描述了一种用于分离、测序和生物信息学分析径向的协议（Radial-Seq）我们期望该分析能够证实 A-EJ 形成这种连接，并极大地促进这种连接的形成。总的来说，这些实验应该扩展我们对径向聚变分子组成的理解。机械地定义径向形成的要求，这应该有利于基础研究人员； A-EJ 的形成可能有助于治疗性地阻碍其形成。