Characterization of Dbf4/Cdc7 function in DNA replication and cell cycle checkpoint responses

DNA 复制和细胞周期检查点反应中 Dbf4/Cdc7 功能的表征

• 批准号: RGPIN-2020-04666
• 负责人: Duncker, Bernard
• 金额: $ 2.62万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740107
• 关键词: Characterization; Dbf4; Cdc7; function; DNA

We live in a time of uncertainty when changes to the environment as a consequence of increased industrial activities pose new risks to all living organisms. Among the most concerning of these is an increased incidence of cellular perturbations. For example, exposure to common manufacturing chemical benzene or to certain types of commercial nanoparticles can lead to DNA damage and genomic instability. My lab is studying the way that cells normally copy their DNA as they divide. Detailed knowledge of how this occurs under normal conditions helps us to understand how things go wrong and leads to new strategies to help prevent harmful outcomes. One of the safeguards cells use to protect their genomes is a surveillance system called cell cycle checkpoints. They monitor the presence of DNA damage, coordinate repair processes when necessary, and eliminate severely damaged cells from the population if they cannot be efficiently restored. We are particularly interested in a key protein called Dbf4, found in all eukaryotic (with a clearly defined nucleus) organisms, which is both required for proper DNA replication to initiate, and is inhibited by checkpoints in response to DNA damage as a means of slowing down DNA replication until the damage has been repaired. The three objectives of my research proposal help us to understand how Dbf4 functions and is regulated. All of the work will be carried out in budding yeast cells, a very useful model organism, with most cellular functions similar to that of other eukaryotic organisms, including humans. For Objective 1 we are proposing to further characterize a newly identified way that a key checkpoint protein, Rad53, interacts with Dbf4. We are interested in determining whether this is specific to the Dbf4-Rad53 interaction, or if it may operate in other interactions involving checkpoint proteins. For Objective 2 we will study how Dbf4 interacts with and opposes the activity a protein called Rif1 which acts as an inhibitor of DNA replication. Of particular interest is a better characterization of the effects of disrupting the Dbf4-Rif1 association, which causes cells to become acutely sensitive to DNA double strand breaks which can occur when cells are exposed to environmental carcinogens. For Objective 3 we will further characterize the interaction between Dbf4 and a protein called Mrc1, which appears to be important for the proper timing of DNA replication. The students involved in this research will benefit from training in a wide range of approaches that they will employ to investigate these aspects of Dbf4 activity and regulation, including biochemistry, molecular biology, genetics, structural analysis and bioinformatic techniques.

我们生活在一个充满不确定性的时代，工业活动增加导致的环境变化给所有生物体带来新的风险。其中最令人担忧的是细胞扰动发生率的增加。例如，接触常见的制造化学品苯或某些类型的商业纳米粒子可能会导致 DNA 损伤和基因组不稳定。我的实验室正在研究细胞分裂时复制 DNA 的方式。详细了解正常情况下这种情况是如何发生的，有助于我们了解事情是如何出错的，并制定新的策略来帮助防止有害结果。细胞用来保护其基因组的保障措施之一是称为细胞周期检查点的监视系统。它们监测 DNA 损伤的存在，必要时协调修复过程，并在无法有效修复的情况下从群体中消除严重受损的细胞。我们对一种名为 Dbf4 的关键蛋白特别感兴趣，它存在于所有真核生物（具有明确定义的细胞核）中，它既是正确 DNA 复制启动所必需的，又被检查点抑制以响应 DNA 损伤，作为减缓 DNA 损伤的一种手段。降低DNA复制直至损伤被修复。我的研究计划的三个目标帮助我们了解 Dbf4 的功能和监管方式。所有的工作都将在出芽酵母细胞中进行，这是一种非常有用的模型生物，其大多数细胞功能与包括人类在内的其他真核生物相似。对于目标 1，我们建议进一步表征关键检查点蛋白 Rad53 与 Dbf4 相互作用的新发现方式。我们感兴趣的是确定这是否是 Dbf4-Rad53 相互作用所特有的，或者它是否可能在涉及检查点蛋白的其他相互作用中发挥作用。对于目标 2，我们将研究 Dbf4 如何与 Rif1 蛋白（作为 DNA 复制抑制剂）的活性相互作用并对抗该蛋白的活性。特别令人感兴趣的是更好地表征破坏 Dbf4-Rif1 关联的影响，这种关联会导致细胞对 DNA 双链断裂变得非常敏感，当细胞暴露于环境致癌物时可能会发生这种情况。对于目标 3，我们将进一步表征 Dbf4 和一种名为 Mrc1 的蛋白质之间的相互作用，该蛋白质对于 DNA 复制的正确计时似乎很重要。参与这项研究的学生将受益于各种方法的培训，他们将使用这些方法来研究 Dbf4 活性和调控的这些方面，包括生物化学、分子生物学、遗传学、结构分析和生物信息学技术。