Mechanistic insight into genome stability pathways

对基因组稳定性途径的机制洞察

• 批准号: 10402940
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 11.27万
• 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 2022-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402940
• 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.

项目概要 基因组完整性取决于强大的 DNA 复制程序和复制耦合修复活动 在细胞周期的不同阶段起作用的途径。我的实验室长期以来一直对 复制压力的原因和后果。当基因组损伤持续存在时，就会出现复制压力 由于修复缺陷或复制机器的组件出现功能障碍。虽然疾病—— 引起必需复制因子突变的情况很少见，它们可以引起多效性和严重的疾病，例如 如免疫缺陷、心肌病或生长缺陷。近年来，我们研究了分子 这些罕见疾病的机制。我们已经鉴定出复合杂合患者突变 复制因子微型染色体维持蛋白 10 (MCM10)，并在人体中对其进行了建模 体细胞系。尽管这些突变导致相对轻微的细胞复制缺陷，但它们构成了 端粒维持的重大问题。当前细胞模型的一个警告是它们是永生的 并组成型表达端粒酶。为了更好地理解复制缺陷的影响 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 并在体外将其分化为特定的细胞类型。这为动物模型提供了一种有价值的替代方案， 相应地，不要完全模仿人类的端粒稳态。此外，我们对以下途径感兴趣： 细胞在轻度复制应激条件下激活生存。之前的工作已经确定了一种基于网络的 泛素化和 SUMO 化，以及环指蛋白 4 (RNF4) 作为关键成分。 RNF4 是一个 SUMO- 靶向 E3 泛素连接酶与双链断裂修复有关，但其在复制中的作用 分叉和端粒维护尚不清楚。基因相互作用筛选已鉴定出布鲁姆 解旋酶 (BLM)，一种导致过早衰老的 RecQ 家族解旋酶，以及泛素特异性肽酶 7 (USP7)， 去泛素酶，作为强负相互作用物。 USP7 的突变与罕见的 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 端粒酶阳性和阴性细胞染色体遗传中的 BLM。最后，还有一个共同的特点 复制压力是由于无法复制整个基因组而导致的复制不足。结果，单 搁浅的缺口仍然存在，可以通过复制后修复来填补，而复制后修复是由泛素化调节的 PCNA 或作为最后的手段，通过有丝分裂 DNA 合成 (MiDAS)。 MiDAS 是一种类似断裂诱导复制 (BIR) 的 与经典的复制叉不同，该途径通过置换合成来复制 DNA。我们将研究如何 泛素化 PCNA 控制 MiDAS，并将确定其他 BIR 相关通路是否受 PCNA 泛素化。总之，本提案中提出的问题将阐明基本的和 人类细胞中基因组稳定性途径的疾病相关机制。