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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717247
• 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.

当增加工业活动会给所有生物体带来新的风险，因此我们生活在不确定性的时代。其中最令人关注的是细胞扰动的发生率增加。例如，暴露于常见的制造化学苯或某些类型的商业纳米颗粒会导致DNA损伤和基因组不稳定性。我的实验室正在研究细胞在分裂时通常复制其DNA的方式。详细了解这种情况如何在正常条件下发生的情况有助于我们了解事情如何出现问题，并导致新的策略，以帮助防止有害结果。细胞用来保护其基因组的保障措施之一是一种称为细胞周期检查点的监视系统。他们监测DNA损伤的存在，必要时协调维修过程，并在无法有效恢复的情况下消除人群中严重受损的细胞。我们对一种称为DBF4的关键蛋白质特别感兴趣，该蛋白在所有真核（具有明确定义的核）生物中发现，这既需要适当的DNA复制才能启动，并且被检查点抑制了DNA损伤，以抑制DNA损伤，作为减慢DNA复制的方法，直到修复了损伤。我的研究建议的三个目标有助于我们了解DBF4的功能和受监管。所有工作都将在萌芽的酵母细胞中进行，这是一种非常有用的模型生物体，其大多数细胞功能与包括人类在内的其他真核生物生物相似。对于目标1，我们建议进一步表征一种新确定的方法，即键检查点蛋白RAD53与DBF4相互作用。我们有兴趣确定这是否特定于DBF4-RAD53相互作用，还是在涉及检查点蛋白的其他相互作用中运行。对于目标2，我们将研究DBF4如何与称为RIF1的蛋白质相互作用并反对活性，该蛋白充当DNA复制的抑制剂。特别令人感兴趣的是，更好地表征了破坏DBF4-RIF1关联的作用，这会导致细胞对DNA双链断裂的敏感性敏感，而DNA双链断裂可能会在细胞暴露于环境致癌物中。对于目标3，我们将进一步表征DBF4与称为MRC1的蛋白质之间的相互作用，这对于DNA复制的适当时机似乎很重要。参与这项研究的学生将受益于他们将采用多种方法来研究DBF4活动和调节的这些方面的培训，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。