The Role of the F-box Protein Dia2 in Cell Cycle Control

描述(申请人提供)：单个细胞的复制涉及一系列严格控制的阶段，称为细胞周期。遗传信息的忠实复制对细胞分裂至关重要，因为DMA复制的中断会导致基因组不稳定，这是癌细胞的一个标志。泛素化是细胞周期进程的关键调节因子之一。高度保守的模块化SCF(Skp1/CDc53/F-box蛋白)泛素连接酶复合体是泛素依赖的细胞分裂调控的重要介体。F-box蛋白是SCF复合体的底物特异性适配子。在人类和模型真核系统中都发现了许多F-box蛋白，SCF家族成员的突变与肿瘤的发生有关。这表明，泛素介导的细胞周期关键蛋白的破坏是控制细胞增殖的重要机制。这项建议的具体目标是确定F-box蛋白Dia2在调控DMA复制中的作用。Dia2是一种染色质结合蛋白，以细胞周期依赖的方式结合复制起点。初步数据表明，Dia2与DNA复制蛋白形成复合体，包括MCM2-7复制型解旋酶和DNA聚合酶a/起始酶复合体的亚单位CDc17。Dia2蛋白的目标是在G1期破坏，这是复制复合体组装的时候。有趣的是，dia2缺失突变体和F-box缺失突变体对DMA损伤剂高度敏感，并积累了DMA损伤焦点，这表明SCFDia2复合体在维持基因组稳定性方面发挥了作用。中心假设是Dia2作为调控DNA复制的SCF泛素连接酶的一部分发挥作用。在缺乏这种功能的情况下，基因组就不稳定。此外，Dia2蛋白的蛋白降解可能在DNA复制的调节中作为开关来控制SCFDia2的活性。这个建议的具体目标是：1.通过确定Dia2如何被招募到复制起始点以及如何控制Dia2的蛋白分解来研究Dia2的调控及其对DNA复制的影响，以及2.通过确定Dia2蛋白复合体的组成并检查它们是否受细胞周期调控来确定Dia2蛋白复合体在DNA复制中的作用。SCFDia2蛋白靶标将通过候选方法和分离底物的遗传方法的持续发展来确定。相关性：该项目研究控制正常细胞生长和增殖的生化过程。当缺陷时，这些过程有助于肿瘤的形成。我们的目标是了解这一过程在正常细胞中是如何工作的，以便在未来开发抗癌疗法。

DESCRIPTION (provided by applicant): The duplication of a single cell involves a series of tightly controlled stages called the cell cycle. The faithful replication of genetic information is essential to cell division as disruption of DMA replication leads to genomic instability, a hallmark of cancer cells. Ubiquitination is one of the key regulators of cell cycle progression. Members of the highly conserved, modular SCF (Skp1/Cdc53/F-box protein) ubiquitin ligase complexes are important mediators of ubiquitin-dependent regulation of cell division. F-box proteins serve as substrate-specific adaptors for SCF complexes. Many F-box proteins have been identified in both humans and model eukaryotic systems and mutation of SCF family members contributes to tumorigenesis. This implies that ubiquitin-mediated destruction of key cell cycle proteins is an important mechanism for controlling cell proliferation. The specific objective of this proposal is to determine the role of the F-box protein Dia2 in the regulation of DMA replication. Dia2 is a chromatin-bound protein that binds replication origins in a cell cycle dependent manner. Preliminary data indicate that Dia2 forms complexes with DMA replication proteins, including the MCM2-7 replicative helicase and Cdc17, a subunit of DMA polymerase a/primase complex. The Dia2 protein is targeted for destruction during G1, a time when replication complexes are assembled. Interestingly, dia2 null and F-box deletion mutants are hypersensitive to DMA damaging agents and accumulate DMA damage foci, suggesting that a SCFDia2 complex plays a role in maintaining genomic stability. The central hypothesis is that Dia2 functions as part of an SCF ubiquitin ligase that regulates DNA replication. In the absence of this function, the genome is unstable. In addition, proteolysis of the Dia2 protein may act as a switch to control SCFDia2 activity in the regulation of DNA replication. The specific goals of this proposal are to: 1. Examine the regulation of Dia2 and its effects on DNA replication by determining how Dia2 is recruited to replication origins and how Dia2 proteolysis is controlled, and 2. Identify the role of Dia2 protein complexes in DNA replication by determining their composition and examining whether they are cell cycle regulated. SCFDia2 protein targets will be identified by a candidate approach and continued development of a genetic method for isolating substrates. Relevance: This project examines biochemical processes that control normal cell growth and proliferation. When defective, these processes contribute to tumor formation. The goal is to understand how the processes work in normal cells so that anti-cancer therapies can be developed in the future.