Mechanistic insight into genome stability pathways

对基因组稳定性途径的机制洞察

• 批准号: 10763597
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 19.18万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10763597
• 关键词: Mechanistic; insight; into; genome; stability

Project Summary Genome integrity depends on a robust DNA replication program and the activity of replication-coupled repair pathways that operate during different phases of the cell cycle. My laboratory has had a longstanding interest in the causes and consequences of replication stress. Replication stress arises when lesions in the genome persist due to repair deficiencies or when components of the replication machinery are dysfunctional. Although disease- causing mutations in essential replication factors are rare, they can cause pleiotropic and severe disorders, such as immunodeficiency, cardiomyopathy, or growth defects. In recent years, we have investigated the molecular mechanism that underlies these rare diseases. We have identified compound heterozygous patient mutations in the replication factor minichromosome maintenance protein 10 (MCM10), and have modeled them in human somatic cell lines. Although these mutations cause relatively mild cellular replication defects, they pose significant problems to telomere maintenance. One caveat of the current cell models is that they are immortalized and express telomerase constitutively. To better understand the impact of replication defects in the context of cellular development of affected tissues, we propose to engineer genome-edited induced pluripotent stem cells and differentiate them into specific cell types in vitro. This presents a valuable alternative to animal models which, relevantly, do not fully mimic telomere homeostasis in humans. Moreover, we are interested in the pathways that cells activate for survival under conditions of mild replication stress. Previous work has identified a network based on ubiquitination and SUMOylation, and ring finger protein 4 (RNF4) as a key component. RNF4 is a SUMO- targeted E3 ubiquitin ligase that has been implicated in double-strand break repair, however, its role at replication forks and in telomere maintenance is not well understood. A genetic interaction screen has identified Bloom helicase (BLM), a RecQ-family helicase that causes premature aging, and ubiquitin specific peptidase 7 (USP7), a deubiquitinase, as strong negative interactors. Mutations in USP7 have been linked to rare neurodevelopmental disorders, but its cellular action has remained obscure. Interestingly, USP7 and BLM also regulate DNA replication and telomere length. We will investigate the relationship between RNF4, USP7 and BLM in chromosome inheritance in telomerase-positive and -negative cells. Lastly, a common feature of replication stress is under-replication due to an inability to duplicate the entire genome. As a result, single- stranded gaps persist that can either be filled by post-replicative repair that is regulated by the ubiquitination of PCNA or, as a last resort, by mitotic DNA synthesis (MiDAS). MiDAS is a break-induced replication (BIR)-like pathway that, unlike a classical replication fork, copies DNA by displacement synthesis. We will study how ubiquitinated PCNA controls MiDAS, and will determine whether other BIR-related pathways are regulated by PCNA ubiquitination. In summary, the questions addressed in this proposal will elucidate fundamental and disease-relevant mechanisms of genome stability pathways in human cells.

项目摘要 基因组完整性取决于强大的DNA复制程序和复制耦合修复的活性 在细胞周期的不同阶段运行的途径。我的实验室对 复制应力的原因和后果。当基因组中的病变持续存在时，会出现复制应力 由于修复缺陷或复制机械的组件功能失调时。虽然疾病 - 引起基本复制因子的突变很少见，它们会引起多效和严重疾病，例如 作为免疫缺陷，心肌病或生长缺陷。近年来，我们研究了分子 这些罕见疾病的基础的机制。我们已经确定了复合杂合子患者突变 复制因子微小颜色体维持蛋白10（MCM10），并在人类中进行了建模 体细胞系。尽管这些突变导致相对温和的细胞复制缺陷，但它们构成了 端粒维护的重大问题。当前细胞模型的一个警告是它们是永生的 并组成性地表达端粒酶。更好地理解复制缺陷的影响 细胞的生长受影响的组织的发展，我们建议设计基因组编辑的诱导多能干细胞 并将它们区分为体外特定细胞类型。这为动物模型提供了宝贵的替代品 相关，不要完全模仿人类的端粒稳态。而且，我们对 细胞在轻度复制应激条件下激活生存。以前的工作已经确定了基于网络的 在泛素化和sumoylation上，环手指蛋白4（RNF4）作为关键成分。 RNF4是相扑 靶向的E3泛素连接酶与双链断裂维修有关，但是，其在复制中的作用 叉子和端粒维护中尚不清楚。遗传相互作用屏幕已经确定了开花 解旋酶（BLM），一种导致过早衰老的RECQ家庭解旋酶，以及泛素特异性肽酶7（USP7），， 去泛素酶，作为强烈的负相互作用。 USP7中的突变与罕见 神经发育障碍，但其细胞作用仍然晦涩难懂。有趣的是，USP7和BLM也 调节DNA复制和端粒长度。我们将研究RNF4，USP7和 BLM在端粒酶阳性和阴性细胞中的染色体遗传中。最后，一个共同特征的 由于无法复制整个基因组，复制应力的复制不足。结果， 搁浅的差距持续存在，可以通过泛滥的修复后修复来填补。 PCNA或作为最后的手段，通过有丝分裂DNA合成（MIDAS）。 MIDAS是突破引起的复制（BIR） 与经典复制叉不同的途径通过位移合成复制DNA。我们将研究如何 泛素化的PCNA控制MIDAS，并将确定其他与BIR相关的途径是否受到调节 PCNA泛素化。总而言之，本提案中提出的问题将阐明基本和 人类细胞中基因组稳定途径相关的疾病机制。