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

我们生活在一个充满不确定性的时代，工业活动增加造成的环境变化给所有生物体带来了新的风险。其中最令人担忧的是细胞扰动的发生率增加。例如，暴露于常见的制造化学品苯或某些类型的商业纳米颗粒可能导致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。详细了解在正常情况下这是如何发生的，有助于我们了解事情是如何出错的，并导致新的策略来帮助防止有害的结果。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，在必要时协调修复过程，并在无法有效恢复时从群体中消除严重受损的细胞。我们特别感兴趣的是一种名为Dbf 4的关键蛋白，它存在于所有真核生物（具有明确定义的细胞核）中，它是启动正确DNA复制所必需的，并且在DNA损伤时被检查点抑制，作为减缓DNA复制的一种手段，直到损伤被修复。我的研究计划的三个目标帮助我们了解Dbf 4的功能和调节方式。所有的工作都将在芽殖酵母细胞中进行，芽殖酵母细胞是一种非常有用的模式生物，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一种新发现的关键检查点蛋白Rad 53与Dbf 4相互作用的方式。我们感兴趣的是确定这是否是特定于Dbf 4-Rad 53相互作用，或者它是否可以在涉及检查点蛋白的其他相互作用中起作用。对于目标2，我们将研究Dbf 4如何与一种称为Rif 1的蛋白质相互作用并对抗其活性，Rif 1作为DNA复制的抑制剂。特别令人感兴趣的是更好地表征破坏Dbf 4-Rif 1关联的影响，这导致细胞对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发生这种情况。对于目标3，我们将进一步表征Dbf 4和一种名为Mrc 1的蛋白质之间的相互作用，该蛋白质似乎对DNA复制的适当时机很重要。参与这项研究的学生将受益于广泛的方法培训，他们将采用这些方法来研究Dbf 4活性和调节的这些方面，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。

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.