Mechanistic insight into genome stability pathways

项目摘要 基因组的完整性依赖于一个强大的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作的途径。我的实验室一直对 复制压力的原因和后果。当基因组中的损伤持续存在时， 由于修复缺陷或当复制机器的组件功能失调时。虽然疾病- 引起基本复制因子突变的基因很少，它们可以引起多效性和严重的疾病， 免疫缺陷心肌病或生长缺陷近年来，我们研究了 这些罕见疾病的潜在机制。我们已经确定了复合杂合子患者突变， 复制因子微小染色体维持蛋白10（MCM 10），并在人体内模拟它们 体细胞系虽然这些突变导致相对轻微的细胞复制缺陷，但它们构成了 对端粒的维护有很大的影响目前的细胞模型的一个警告是，它们是永生的 并组成性表达端粒酶。为了更好地了解复制缺陷在 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成特定的细胞类型。这为动物模型提供了一种有价值的替代方法， 相关地，不能完全模拟人类的端粒稳态。此外，我们感兴趣的途径， 细胞在轻度复制应激条件下活化存活。以前的工作已经确定了一个基于网络的 环指蛋白4（Ringfingerprotein 4，RNF 4）是泛素化和SUMO化的关键组分。RNF 4是一种相扑- 靶向E3泛素连接酶，已牵连在双链断裂修复，然而，其作用在复制 分叉和端粒的维护还不是很清楚。基因交互筛选已经确定布鲁姆 解旋酶（BLM），一种导致过早衰老的RecQ家族解旋酶，和泛素特异性肽酶7（USP 7）， 一种去泛素化酶，作为强负相互作用物。USP 7的突变与罕见的 神经发育障碍，但其细胞作用仍然不清楚。有趣的是，USP 7和BLM也 调节DNA复制和端粒长度。我们将研究RNF 4，USP 7和 BLM在端粒酶阳性和阴性细胞染色体遗传中的作用。最后，一个共同的特点是， 复制应激是由于不能复制整个基因组而导致的复制不足。因此，单- 滞留的缺口持续存在，可以通过复制后修复来填补，这是由泛素化调节的， PCNA或作为最后手段，通过有丝分裂DNA合成（MiDAS）。MiDAS是一种断裂诱导复制（BIR）样 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 泛素化的PCNA控制MiDAS，并将决定其他BIR相关途径是否受 PCNA泛素化。总而言之，本提案中所涉及的问题将阐明基本的 人类细胞中基因组稳定性途径的疾病相关机制。

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.