Mechanism of radial chromosome formation in human premature aging syndrome cells

项目总结 50多年来，放射状染色体(以下简称“放射状”)一直是科学意识的一部分。 好几年了。放射状染色体在操作上被定义为涉及两个或更多非同源染色体的融合 有不止一个着丝粒。在正常细胞中，放射状细胞相当罕见，而且几乎从未形成过。 自然而然地。然而，放射状血管具有重要的临床意义，因为它们的出现是一种诊断。 骨髓衰竭、癌症易感性和早衰综合征、范可尼贫血的特征 (FA)。有趣的是，即使在FA细胞中，放射状染色体也不会自发地出现高水平的 频率，但通常需要通过接触临床相关的DNA交联剂来诱导，例如 丝裂霉素C(MMC)和顺铂。尽管它们的诊断使用了几十年，但径向地层仍然很差。 理解，文献中充满了关于他们的机械需求的相互矛盾的报告 制作。在我们的第一个特定目标中，我们描述了构建一个具有缺陷的人类细胞系 辐射敏感18(RAD18)基因。RAD18编码E3泛素连接酶，是跨损伤所必需的 合成(TLS)，一个容易出错的DNA损伤耐受途径，是FA介导的一个子途径 DNA交联链修复过程。与这一作用相一致的是，RAD18缺失的人类细胞产生了高水平的MMC- 诱导径向。出乎意料的是，DNA连接酶III(LIGIII)和DNA聚合酶Theta(POLQ；两者)的缺失 替代非同源末端连接的关键成分；A-EJ))显著减少了对RAD18的依赖， MMC诱导的放射状地层。因此，由于没有RAD18而产生的径向似乎需要A- EJ途径。我们建议证实这些观察结果，并通过以下方式扩展这些研究的一般性 丝裂霉素诱导的Bloom综合征(BLM)放射状结构对A-EJ需求的确定(另一种 癌症易感性和早衰综合征)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。 染色体融合点。这是不幸的，因为对各种其他生物的融合连接的分析 易位类型(例如，典型的染色体相互易位和端粒融合)提供了 关于这种连接的分子特征的丰富数据以及对修复的洞察 负责它们形成的机制。在我们的具体目标2中，我们建议纠正这一严重的遗漏。 具体地说，我们描述了一种用于分离、测序和生物信息分析RADIUS的方法(Radial-Seq 染色体融合。我们期望这项分析将证实A-EJ形成这种连接，并在很大程度上 扩展我们对径向聚变的分子组成的理解。总之，这些实验应该 从机械上定义有利于基础研究人员的径向地层要求；此外， 为它们的形成分配A-EJ可能有助于临床医生从治疗上阻碍它们的形成。

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.