Mechanistic insight into genome stability pathways

对基因组稳定性途径的机制洞察

• 批准号: 10205825
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 30.25万
• 依托单位: UNIVERSITY OF MINNESOTA
• 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/10205825
• 关键词: Address; Affect; Animal Model; Cardiomyopathies; Cell Cycle; Cell Line; Cell model; Cells; Chromosomes; Coupled; DNA; DNA biosynthesis; DNA replication fork; Defect; Development; Disease; Double Strand Break Repair; Family; Genetic; Genome; Genome Stability; Genome engineering; Growth; Homeostasis; Human; Immune system; Immunologic Deficiency Syndromes; In Vitro; Laboratories; Length; Lesion; Link; Maintenance; Malignant Neoplasms; Mitotic; Modeling; Molecular; Mutation; Network-based; Neurodevelopmental Disorder; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Phase; Premature aging syndrome; Proteins; Rare Diseases; Resort; Ring Finger Domain; Role; Somatic Cell; Sumoylation Pathway; Telomerase; Telomere Maintenance; Tissues; Ubiquitin; Ubiquitination; Work; cell type; cellular development; disease-causing mutation; genome editing; genome integrity; helicase; human disease; induced pluripotent stem cell; insight; interest; novel diagnostics; novel therapeutics; programs; repaired; replication stress; stem cell differentiation; telomere; ubiquitin-protein ligase; whole genome

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 基因组的完整性依赖于强大的DNA复制程序和复制偶联修复的活性 在细胞周期的不同阶段运行的通路。我的实验室长期以来一直对 复制应激的原因和后果。当基因组中的损伤持续存在时，复制压力就会出现 由于修复缺陷或当复制机械的部件出现故障时。尽管疾病- 导致基本复制因子突变的情况很少见，它们可能导致多效性和严重的疾病，如 如免疫缺陷、心肌病或生长缺陷。近几年来，我们一直在研究分子 这些罕见疾病背后的机制。我们已经确定了患者体内的复合杂合子突变 复制因子微小染色体维持蛋白10(MCM10)，并在人体内模拟它们 体细胞系。尽管这些突变会导致相对轻微的细胞复制缺陷，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不朽的 并结构性地表达端粒酶。为了更好地了解复制缺陷在以下环境中的影响 对于受影响组织的细胞发育，我们建议设计基因组编辑的诱导多能干细胞 并在体外将它们分化为特定的细胞类型。这为动物模型提供了一个有价值的替代方案， 相应地，不要完全模仿人类的端粒稳态。此外，我们感兴趣的是 在温和的复制压力条件下，细胞被激活以求生存。以前的工作已经确定了一个基于 关于泛素化和SuMO化，以及环指蛋白4(RNF4)作为关键成分。RNF4是一场相扑- 靶向E3泛素连接酶参与双链断裂修复，然而，它在复制中的作用 叉子和端粒的维持还不是很清楚。一个基因互动屏幕发现了布鲁姆 解旋酶(BLM)，一种导致过早衰老的RecQ家族解旋酶，以及泛素特异肽酶7(USP7)， 一种脱泛素酶，作为强大的负相互作用。USP7基因突变与罕见的 神经发育障碍，但其细胞作用仍不清楚。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染色体遗传中的作用最后，它的一个共同特点是 复制压力是由于无法复制整个基因组而导致复制不足。因此，单一的- 滞留的缺口持续存在，要么可以通过复制后修复来填补，后者由泛素化的 增殖细胞核抗原，或作为最后手段，通过有丝分裂DNA合成(MIDAS)。MIDAS是类似于中断诱导复制(BIR)的 与经典的复制分叉不同的是，通过置换合成复制DNA的途径。我们将研究如何 泛素化的增殖细胞核抗原控制MIDAS，并将决定其他与BIR相关的通路是否受 增殖细胞核抗原泛素化。总而言之，本提案中涉及的问题将阐明基本的和 人类细胞基因组稳定性途径的疾病相关机制。