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

我们生活在一个不确定的时代，工业活动增加导致的环境变化给所有生物带来了新的风险。其中最令人担忧的是细胞扰动的发生率增加。例如，接触普通制造化学品苯或某些类型的商业纳米颗粒可能会导致DNA损伤和基因组不稳定。我的实验室正在研究细胞分裂时正常复制DNA的方式。详细了解这种情况在正常情况下是如何发生的，有助于我们理解事情是如何出错的，并导致帮助防止有害后果的新战略。细胞用来保护自己基因组的安全措施之一是一个被称为细胞周期检查点的监测系统。他们监测DNA损伤的存在，在必要时协调修复过程，并在无法有效恢复的情况下从种群中消除严重受损的细胞。我们特别感兴趣的是一种名为Dbf4的关键蛋白，它存在于所有真核生物(具有明确定义的核)中，它既是启动正常DNA复制所必需的，也是响应DNA损伤的检查点所抑制的，作为一种减缓DNA复制的手段，直到损伤得到修复。我的研究提案的三个目标帮助我们了解DBF4是如何发挥作用和受到监管的。所有的工作都将在发芽酵母细胞中进行，这是一种非常有用的模式生物，其大部分细胞功能与包括人类在内的其他真核生物的功能相似。对于目标1，我们建议进一步描述一种新发现的关键检查点蛋白Rad53与Dbf4相互作用的方式。我们感兴趣的是确定这是否是Dbf4-Rad53相互作用的特异性，或者它是否可能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Dbf4如何与一种名为Rif1的蛋白质相互作用和对抗这种活性，Rif1是一种DNA复制抑制因子。特别令人感兴趣的是更好地描述了破坏Dbf4-Rif1结合的影响，这导致细胞对DNA双链断裂变得非常敏感，当细胞暴露在环境致癌物中时就会发生这种情况。对于目标3，我们将进一步描述Dbf4与一种名为MRc1的蛋白质之间的相互作用，这似乎对DNA复制的适当时机很重要。参与这项研究的学生将从广泛的方法培训中受益，他们将使用这些方法来研究Dbf4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.