The role of ubiquitin-like proteins (UBLs) in DNA repair in vertebrates

类泛素蛋白 (UBL) 在脊椎动物 DNA 修复中的作用

• 批准号: RGPIN-2015-05616
• 负责人: Dellaire, Graham
• 金额: $ 2.48万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613649
• 关键词: role; ubiquitin; like; proteins; UBLs

The ability to faithfully repair DNA damage is a fundamental property of all living organisms. The major aim of my NSERC research program is to uncover how DNA damage is detected, the signaling and factors that determine DNA repair pathway choice between error-prone or error-free mechanisms, and how DNA repair proceeds in vivo within our cells. To date, DNA repair has been studied primarily in cultured cells grown as a single cell layer. While valuable information has been gained, this approach does not fully represent how our cells grow in 3 dimensions, interact with other cells in a tissue,  divide and respond to DNA damage inside our bodies. A number of animal model systems, including worms, flies and more recently, zebrafish, have been used to study biological processes in vivo in whole organisms. Zebrafish are an attractive animal model because unlike worms or flies, they are vertebrates and share a high degree of genetic similarity with humans. Furthermore, zebrafish embryos are completely transparent allowing complex biological processes like DNA repair to be observed in cells within the living animal by fluorescence microscopy. We will use cutting-edge genome engineering and molecular genetic techniques to create zebrafish with altered expression of key DNA repair proteins, or that express fluorescent versions of these proteins, allowing DNA repair to be observed for the first time in a living vertebrate in “real time” by fluorescence microscopy. Using these transgenic zebrafish as our model system, we will investigate several members of a class of proteins known as ubiquitin-like modifiers (UBLs) for their role in DNA repair, which remains poorly defined. In complementary studies, we will use biochemical approaches, mass spectrometry and protein-protein interaction assays to assess the role of these UBLs in human cells and to identify novel molecular pathways involving UBLs implicated in the DNA damage response, as well as their conservation in zebrafish. This research will expand our knowledge of the fundamental process of DNA repair and the role of UBLs in the DNA damage response. These studies will be among the first to study DNA repair in a living vertebrate animal, and will create new tools for scientists to study the DNA damage response using zebrafish. We anticipate our findings will be of broad interest to geneticists, molecular biologists, biochemists, and biotechnologists using microscopy to study fundamental biology.

忠实地修复DNA损伤的能力是所有生物体的基本属性。我的NSERC研究计划的主要目的是揭示如何检测DNA损伤，决定DNA修复途径在易错或无错机制之间选择的信号和因素，以及DNA修复如何在我们的细胞内进行。迄今为止，DNA修复主要在作为单细胞层生长的培养细胞中进行研究。虽然已经获得了有价值的信息，但这种方法并不能完全代表我们的细胞如何在三维空间中生长，与组织中的其他细胞相互作用，分裂并对我们体内的DNA损伤做出反应。许多动物模型系统，包括蠕虫、苍蝇和最近的斑马鱼，已被用于研究整个生物体内的生物过程。斑马鱼是一种有吸引力的动物模型，因为与蠕虫或苍蝇不同，它们是脊椎动物，与人类具有高度的遗传相似性。此外，斑马鱼胚胎是完全透明的，可以通过荧光显微镜在活体动物的细胞中观察到复杂的生物过程，如DNA修复。我们将使用尖端的基因组工程和分子遗传技术来创造斑马鱼与关键DNA修复蛋白的表达改变，或表达这些蛋白质的荧光版本，允许DNA修复被观察到的第一次在活的脊椎动物在“真实的时间”通过荧光显微镜。使用这些转基因斑马鱼作为我们的模型系统，我们将研究一类蛋白质的几个成员被称为泛素样修饰剂（UBLs），其在DNA修复中的作用，这仍然很难定义。在补充研究中，我们将使用生物化学方法，质谱和蛋白质-蛋白质相互作用测定来评估这些UBL在人类细胞中的作用，并确定涉及DNA损伤反应的UBL的新分子途径，以及它们在斑马鱼中的保护。这项研究将扩大我们对DNA修复的基本过程以及UBL在DNA损伤反应中的作用的认识。这些研究将是第一批研究活体脊椎动物DNA修复的研究，并将为科学家研究斑马鱼的DNA损伤反应创造新的工具。我们预计我们的研究结果将广泛的遗传学家，分子生物学家，生物化学家和生物技术学家使用显微镜研究基础生物学的兴趣。