Studies of the Swi1-Swi3 Replication Fork Protection Complex

描述(申请人提供)：基因组不稳定是各种遗传病的主要原因，最明显的是癌症。为了确保基因组的完整性，细胞必须在每个细胞周期内绝对准确地复制数百万或数十亿个DNA碱基对。然而，细胞一直处于多种因素的压力下，这些因素会导致DNA损伤或阻止DNA复制。为了绕过这些问题，细胞配备了一个质量控制系统，称为DNA复制检查点。在人类中，这个检查点的缺陷会导致遗传不稳定，导致发育和神经缺陷，并极易患癌症。因此，这项应用的长期目标是阐明检查点如何保持基因组的完整性，以防止各种遗传疾病。Swi1-Swi3复合体，也被称为复制叉保护复合体(FPC)，在裂殖酵母中激活DNA复制检查点和稳定停滞的复制叉中起着核心作用。Swi1-Swi3复合体在萌芽酵母中与Tof1-Csm3在进化上是保守的，在人类中与Tof1-Csm3同源。然而，FPC如何稳定复制分叉以及它如何控制复制检查点尚不清楚。因此，第一个目标是了解复制分叉上FPC的分子基础。我们将阐明FPC如何识别复制分叉。第二个目的是确定Swi1磷酸化和DDK-FPC相互作用的功能意义。我们将研究CDC7样激酶复合体在FPC的磷酸化和调节中的作用。第三个目的是了解MRc1-FPC相互作用在分叉稳定和复制检查点激活中的作用。我们将阐明MRc1如何与FPC协同调节其功能，以了解叉子稳定和检查点信号的分子机制。这些研究旨在检验中心假设，即FPC通过与复制分叉以及与DDK和MRc1的相互作用来协调复制分叉的稳定和检查点信号。将在裂殖酵母中进行遗传和生化研究，因为它已被证明是研究细胞周期控制和基因组维持机制的特殊模式系统，这些机制在包括人类在内的真核生物中高度保守。这些实验的成功完成将为指导人类基因组维持机制的研究提供一个更好的框架。项目简介：细胞一直处于阻止DNA复制的许多因素的压力下。这些因素经常导致基因改变，从而导致发育和神经缺陷，以及各种遗传性疾病，最明显的是癌症。因此，阐明细胞如何应对复制抑制对于理解出生缺陷和癌症发展的机制至关重要，有助于治疗药物的开发。

DESCRIPTION (provided by applicant): Genomic instability is a major cause of various genetic disease, most notably cancer. To ensure genomic integrity, cell must replicate the millions or billions of DNA base pairs with absolute fidelity every cell cycle. However, cells are constantly under stress of many factors that cause DNA damage or block DNA replication. To circumvent these problems, cells are equipped with a quality control system, termed the DNA replication checkpoint. In humans, defects in this checkpoint cause genetic instability, leading to developmental and neurological defects, and a strong predisposition to cancer. Therefore, the long-term objective of this application is to elucidate how the checkpoints maintain genomic integrity to prevent a variety of genetic disorders. The Swi1-Swi3 complex, which is also known as the Replication Fork Protection Complex (FPC) , plays a central role in activation of the DNA replication checkpoint and stabilization of stalled replication forks in the fission yeast Schizosaccharomyces pombe. The Swi1-Swi3 complex is evolutionally conserved and homologous to Tof1-Csm3 in budding yeast Saccharomyces cerevisiae and Timeless-Tipin in humans. However, how the FPC stabilizes the replication forks, and how it controls the replication checkpoint, are unknown. Therefore, the first aim is to understand the molecular basis of the FPC at the replication forks. We will elucidate how the FPC recognizes the replication forks. The second aims is to establish the functional significance of Swi1 phosphorylation and DDK-FPC interaction. We will investigate the roles a Cdc7-like kinase complex in phosphorylation and regulation of the FPC. The third aim is to understand the roles of Mrc1-FPC interaction in fork stabilization and activation of the replication checkpoint. We will elucidate how the Mrc1 cooperates with FPC to regulate its function to understand the molecular mechanisms of fork stabilization and checkpoint signaling. These studies are designed to test the central hypothesis that FPC coordinates replication fork stabilization and checkpoint signaling through its association with replication forks and interaction with DDK and Mrc1. Genetic and biochemical studies will be carried out in the fission yeast Schizosaccharomyces pombe because it has been shown to be an exceptional model system for studying cell cycle control and genome maintenance mechanisms which are highly conserved amongst eukaryotes, including humans. Successful completion of these experiments should provide a much better framework for guiding investigations of genome maintenance mechanisms in humans. Project Narrative: Cells are constantly under the stress of many factors that arrest DNA replication. These factors often cause genetic alterations, which lead to developmental and neurological defects, and a variety of genetic diseases, most notably cancer. Therefore, elucidating how cells cope with replication arrest is essential for understanding the mechanisms of birth defects and development of cancer, contributing to the development of therapeutic agents.