Enzymology of Mitosis Promoting Factor (MPF)

真核细胞通过采用检查点监视系统来保持基因组的完整性。这些 调节网络检测细胞核中不完全复制或受损DNA的存在， 从而触发对有丝分裂阻断。在脊椎动物中，检测不完全复制的过程 DNA，被称为DNA复制检查点，涉及上游调节激酶ATR， 下游效应激酶Chk 1。此外，还有一种称为Claspin的关键介导蛋白， 通过ATR调节Chk 1的活化。Claspin是一种Chk 1结合蛋白，直接与DNA结合 复制分叉，似乎是正在进行的DNA合成的传感器。因此，Claspin扮演了一个 在DNA复制检查点中起关键作用。拟议的研究将旨在了解 Claspin的功能和调节。大多数实验将在非洲爪蟾卵提取物中进行， 系统，其中DNA复制检查点的组件可以在无细胞反应中进行研究。 具体的实验目标将包括研究Claspin及其蛋白质-蛋白质之间的修饰 相互作用控制其介导Chk 1激活的能力。结合这些研究， Claspin控制Chk 1激活的分子机制将被探索。值得注意的是，它已经 已经确定Claspin在检查点响应终止后通过一种抑制剂失活。 这个过程叫做适应。消极的潜在机制和更广泛的生理作用 将研究Claspin的调节。最后，Claspin是一种多功能蛋白质的假设， 拥有一个额外的，检查点独立的作用，调节DNA复制叉将是 探讨了这项研究与公共卫生有关，因为癌症是由渐进的 关键调控基因突变的积累。蛋白质如Claspin有助于防止传播 在细胞分裂时突变的基因。因此，拟议的研究将有助于了解 细胞如何避免致癌突变。

Eukaryotic cells preserve the integrity of the genome by employing checkpoint surveillance systems. These regulatory networks detect the presence of incompletely replicated or damaged DNA in the nucleus and thereupon trigger a blockade to mitosis. In vertebrates, the process that detects incompletely replicated DNA, which is known as the DNA replication checkpoint, involves the upstream regulatory kinase ATR and the downstream effector kinase Chk1. In addition, there is a critical mediator protein called Claspin that regulates the activation of Chk1 by ATR. Claspin is a Chk1 -binding protein that associates directly with DNA replication forks and appears to be a sensor of ongoing DNA synthesis. Consequently, Claspin plays a pivotal role in the DNA replication checkpoint. The proposed studies will be aimed at understanding the function and regulation of Claspin. Most of these experiments will be conducted in Xenopus egg extracts, a system in which the components of the DNA replication checkpoint can be studied in a cell-free reaction. Specific experimental objectives will include studies of how modifications of Claspin and its protein-protein interactions control its ability to mediate the activation of Chk1. In conjunction with these studies, the molecular mechanism by which Claspin controls the activation of Chk1 will be explored. Significantly, it has been established that Claspin undergoes inactivation upon termination of a checkpoint response by a process called adaptation. The underlying mechanisms and broader physiological role(s) for the negative regulation of Claspin will be investigated. Finally, the hypothesis that Claspin is a multifunctional protein that possesses an additional, checkpoint-independent role that regulates the DNA replication fork will be explored. This research is relevant to public health because cancer arises from the progressive accumulation of mutations in key regulatory genes. Protein such as Claspin help to prevent the transmission of mutated genes at the time of cell division. Therefore, the proposed studies will aid in the understanding of how cells avoid cancer-inducing mutations.