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

• 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
• 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' 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

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.

项目摘要 放射状染色体（以下简称“放射状”）已经成为科学意识的一部分超过50年 年放射状染色体在操作上被定义为涉及两个或更多个非同源染色体的融合， 有不止一个着丝粒。在正常细胞中，辐射是相当罕见的，他们几乎从来没有形成 自发的然而，桡骨具有重要的临床相关性，因为它们的外观是诊断性的。 骨髓衰竭的标志，癌症易感性和过早衰老综合征，范可尼贫血 （FA）。有趣的是，即使在FA细胞中，放射状染色体也不会自发出现， 但通常需要通过暴露于临床相关的DNA交联试剂来诱导，例如 丝裂霉素C（MMC）和顺铂。尽管放射状地层的诊断应用已有几十年， 理解，文献中充满了关于机械上需要什么的相互矛盾的报告， 生产在我们的第一个具体目标中，我们描述了一种有缺陷的人类细胞系的构建。 辐射敏感18（radiationsensitive 18，RAD 18）基因。RAD 18编码E3泛素连接酶，并且是跨损伤所需的 合成（TLS），一种易错DNA损伤耐受途径，其是FA介导的DNA损伤耐受途径中的子途径。 DNA交联修复过程。与此作用一致，RAD 18-null人类细胞产生高水平的MMC-1。 诱导辐射出乎意料的是，DNA连接酶III（LIGIII）和DNA聚合酶θ（POLQ;两者均 选择性非同源末端连接的关键组分; A-EJ））显著降低了RAD 18依赖性， MMC诱导的放射状形成。因此，由于缺乏RAD 18而产生的辐射似乎需要A- EJ途径。我们建议确认这些观察结果，并扩大这些研究的一般性， 确定在布卢姆综合征（BLM）中MMC诱导的放射状形成中对A-EJ的需要（另一个 癌症易感性和过早衰老综合征）和FA缺陷细胞。 令人惊讶的是，在文献中没有一个关于放射状的分子组成的报道。 染色体融合点这是不幸的，因为对各种各样的其他融合点的分析 易位的类型（例如，典型的相互染色体易位和端粒融合）提供了 大量关于这种连接的分子特征的数据以及对修复的深入了解， 负责其形成的机制。在我们的具体目标2中，我们建议纠正这一令人震惊的遗漏。 具体来说，我们描述了一个协议（径向测序）的分离，测序和生物信息学分析径向 染色体融合我们预计这项分析将证实A-EJ形成这种连接，并大大 扩展了我们对放射状核聚变分子组成的理解总的来说，这些实验应该 机械地定义了对径向形成的要求，这将使基础研究人员受益;此外， 将A-EJ分配用于它们的形成可以帮助临床医生在治疗上阻止它们的形成。