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

F-box 蛋白 Dia2 在细胞周期控制中的作用

• 批准号: 7921277
• 负责人: Deanna M. Koepp
• 金额: $ 12.2万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921277
• 关键词: Binding; Binding Proteins; Biochemical; Biochemical Process; Biological; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cell Proliferation; Cell division; Cells; Chromatin; Color; Complex; DNA Damage; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Development; Exhibits; F Box Domain; F-Box Proteins; Family member; Flow Cytometry; Future; Genetic; Genetic Screening; Genome; Genome Stability; Genomic Instability; Goals; Human; Hypersensitivity; Kinetics; Measures; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Mutation; Nature; Normal Cell; Pathway interactions; Phenotype; Play; Polymerase; Process; Protein Binding; Proteins; Proteolysis; Recruitment Activity; Regulation; Replication Origin; Research; Role; SKP Cullin F-Box Protein Ligases; Saccharomyces cerevisiae; Series; Staging; System; Tertiary Protein Structure; Testing; Time; Ubiquitin; Ubiquitination; Work; base; cancer cell; cancer therapy; cell growth; cell growth regulation; helicase; member; mutant; overexpression; protein complex; protein degradation; tumor; tumorigenesis; ubiquitin ligase

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.

描述（由申请人提供）：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于细胞分裂是必不可少的，因为DMA复制的中断导致基因组不稳定性，这是癌细胞的标志。泛素化是细胞周期进程的关键调节因子之一。高度保守的SCF（Skp 1/Cdc 53/F-box蛋白）泛素连接酶复合物的成员是泛素依赖的细胞分裂调节的重要介质。F-box蛋白作为SCF复合物的底物特异性衔接子。许多F-box蛋白已在人类和模型真核系统中被鉴定，SCF家族成员的突变有助于肿瘤的发生。这意味着遍在蛋白介导的关键细胞周期蛋白的破坏是控制细胞增殖的重要机制。该提案的具体目标是确定F盒蛋白Dia 2在调节DMA复制中的作用。Dia 2是染色质结合蛋白，其以细胞周期依赖性方式结合复制起点。初步数据表明，Dia 2与DMA复制蛋白形成复合物，包括MCM 2 -7复制解旋酶和Cdc 17，DMA聚合酶a/引发酶复合物的亚基。Dia 2蛋白在G1期间被靶向破坏，这是复制复合物组装的时间。有趣的是，dia 2空和F盒缺失突变体对DMA损伤剂超敏感，并积累DMA损伤灶，表明SCFDia 2复合物在维持基因组稳定性中起作用。核心假设是Dia 2作为SCF泛素连接酶的一部分发挥作用，调节DNA复制。如果没有这种功能，基因组是不稳定的。此外，Dia 2蛋白的蛋白水解可以作为开关来控制SCFDia 2在DNA复制调节中的活性。本提案的具体目标是：1.通过确定Dia 2如何被招募到复制起点以及Dia 2蛋白水解如何被控制来检查Dia 2的调节及其对DNA复制的影响，以及2.通过确定Dia 2蛋白复合物的组成并检查它们是否受细胞周期调节，确定Dia 2蛋白复合物在DNA复制中的作用。SCFDia 2蛋白靶点将通过候选方法进行鉴定，并继续开发用于分离底物的遗传方法。相关性：该项目研究控制正常细胞生长和增殖的生化过程。当有缺陷时，这些过程有助于肿瘤形成。我们的目标是了解这些过程在正常细胞中是如何工作的，以便将来可以开发抗癌疗法。