Defining Genome Stability Mechanisms and their Regulation by SUMO and Ubiquitin

保持遗传完整性对于细胞存活和疾病抑制至关重要。因此，蜂窝 DNA损伤反应(DDR)等机制已经演变为对抗持续的遗传毒性侮辱。 虽然已经非常详细地定义了特定的DNA修复机制，但理解时空 对细胞内DDR因子的调节仍然是一个关键挑战。这里，共价翻译后修饰语 (PTMS)相扑和泛素起着关键作用，它们都将DDR蛋白募集到DNA损伤处，然后移除 如果有必要，它们将促进修复。事实上，相扑和泛素途径的缺陷导致了DDR失败 协调，严重的遗传不稳定，和疾病。因此，我们研究的首要目标是 描述相扑和泛素介导的维持基因组完整性的机制，着眼于识别 以及开发潜在的治疗途径。我们的建议以两个因素为中心，STUbL和SMC5/6，这两个因素 通过相扑和泛素整合信号，以支持与健康相关的关键过程。值得注意的是，STUbL 介导三氧化二砷对白血病的治疗作用，SMC5/6突变导致严重疾病。 STUbL是一种E3泛素连接酶，选择性识别和泛素化Sumoylated蛋白以促进 它们的降解和/或从染色质中提取。SMC5/6在功能上与凝集素和凝集素有关 但独一无二的是，可以用相扑和泛素来修改目标。为了提供功能洞察，我们使用了邻近性 标记以可靠地定位STUbL和SMC5/6在关键健康相关环境中的蛋白质组环境 AS：功能失调的端粒、DNA修复和病毒复制。令人惊讶的是，发现了相当大的重叠 在每个蛋白质组之间，在我们的研究中创造进一步的协同效应和效率。例如，函数 并且SMC5/6和STUbL的靶点相交于使用的“交替延长端粒”(ALT)途径 在约15%的癌症中。因此，我们将定义STUbL和SMC5/6在伸长和修剪中的角色 端粒通过我们确定的新的ALT特异性靶点和辅助因子。此外，最近的一大批 数据支持我们的假设，即STUbL控制相扑途径的动态平衡，以及特定的靶点，以 支持基因组稳定、DNA复制和细胞存活。利用遗传操作进行进一步分析 相扑途径(如CRISPR/Cas9)、相扑毒性的介体和新的STUbL靶标将 在相扑和泛素途径串扰中建立这一关键范式。我们最近还发现了SMC5/6 结合相扑并将复合体定向到相分离的ALT PML核体、病毒部位的辅因子 复制，以及可能的DNA损伤，从而统一了这些看似不同的过程。因此，我们会 定义SMC5/6及其新的相扑结合辅因子在每个过程中的功能，以揭示共同的 机械装置。总体而言，我们的协作团队使用蛋白质组、遗传学、细胞分析STUbL和SMC5/6 生物、生化和生物物理方法将协同确定与健康有关的关键机制 相扑和泛素途径的联系；为治疗干预提供目标和指导。

Maintaining genetic integrity is crucial for cell viability and disease suppression. Consequently, cellular machinery such as the DNA damage response (DDR) has evolved to combat continual genotoxic insults. Whilst specific DNA repair mechanisms have been defined in great detail, understanding the spatiotemporal regulation of DDR factors in the cell remains a key challenge. Here, the covalent postranslational modifiers (PTMs) SUMO and ubiquitin play critical roles, both recruiting DDR proteins to DNA lesions, and then removing them as necessary to promote repair. Indeed, defects in the SUMO and ubiquitin pathways cause failed DDR orchestration, severe genetic instability, and disease. Therefore, the overarching goal of our research is to delineate SUMO and ubiquitin mediated mechanisms that maintain genome integrity, with an eye to identifying and exploiting potential therapeutic avenues. Our proposal centers on two factors, STUbL and SMC5/6, which integrate signaling through SUMO and ubiquitin to support key health-related processes. Of note, STUbL mediates the therapeutic effects of arsenic trioxide in leukemia, and SMC5/6 mutations cause severe disease. STUbL is an E3 ubiquitin ligase that selectively recognizes and ubiquitinates SUMOylated proteins to promote their degradation and/or extraction from chromatin. SMC5/6 is functionally related to cohesin and condensin but uniquely, can modify targets with SUMO and ubiquitin. To provide functional insights, we used proximity labeling to reliably map the proteomic environments of STUbL and SMC5/6 in key health-related settings such as: dysfunctional telomeres, DNA repair, and viral replication. Surprisingly, considerable overlap was identified between each proteome, creating further synergy and efficiency in our research. For example, the functions and targets of SMC5/6 and STUbL intersect in the “alternative lengthening of telomeres” (ALT) pathway used in ~15% of cancers. Thus, we would define STUbL and SMC5/6 roles in the elongation and “trimming” of telomeres through the novel ALT-specific targets and cofactors we identified. In addition, a wealth of recent data supports our hypothesis that STUbL controls SUMO pathway homeostasis, as well as specific targets, to support genome stability, DNA replication, and cell survival. Further analysis using genetic manipulation of the SUMO pathway (e.g. CRISPR/Cas9), mediators of SUMO chain toxicity, and new STUbL targets would establish this key paradigm in SUMO and ubiquitin pathway crosstalk. We also recently identified an SMC5/6 cofactor that binds SUMO and directs the complex to phase-separated ALT PML nuclear bodies, sites of viral replication, and likely DNA lesions, thereby unifying these seemingly disparate processes. Hence, we would define functions for SMC5/6 and its new SUMO binding cofactor in each of these processes to reveal common mechanisms. Overall, our collaborative teams' analysis of STUbL and SMC5/6 using proteomic, genetic, cell biological, biochemical, and biophysical methods would synergize to define key health-related mechanisms at the nexus of the SUMO and ubiquitin pathways; providing targets and guidance for therapeutic interventions.