Linking DNA Replication Origin Licensing with Cell Cycle Progression

将 DNA 复制起点许可与细胞周期进展联系起来

• 批准号: 8829782
• 负责人: Kate E Coleman
• 金额: $ 2.49万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8829782
• 关键词: Address; Adult; Apoptosis; Binding; Biological Markers; Biotinylation; Bypass; Cell Cycle; Cell Cycle Kinetics; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cells; Cellular Stress Response; Chromatin; Chromosomes; Competence; Complex; Cues; DNA biosynthesis; DNA replication origin; Defect; Dependence; Development; Diagnostic; Equilibrium; Event; Flow Cytometry; Future; G1 Arrest; G1 Phase; G1/S Transition; G2 Phase; Genome; Genomic Instability; Goals; Health; Human; Knowledge; Lead; Licensing; Licensing Factor; Link; Liver; MAP Kinase Gene; MAPK14 gene; MAPK8 gene; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Mitosis; Molecular; Mutation; Normal Cell; Nuclear; Pattern; Phase Transition; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Process; Proliferating; Proteins; Reagent; Regulation; Replication Initiation; Replication Licensing; Repression; Research; Role; S Phase; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Stomach; Streptavidin; Stress; Testing; Tissues; Tumor-Derived; Work; anaphase-promoting complex; anti-cancer therapeutic; base; cancer cell; cancer diagnosis; cancer therapy; cell transformation; comparative; cooking; extracellular; genome integrity; helicase; in vivo; insight; mitogen-activated protein kinase p38; neoplastic cell; new therapeutic target; novel; novel therapeutics; overexpression; prevent; research study; therapeutic development; therapy development; tool; tumorigenesis

DESCRIPTION (provided by applicant): To maintain proper genome integrity, it is critical that cells properly coordinate intracellular and extracellular signaling pathways controlling DNA replication. Improper regulation of this fundamental process can ultimately contribute to inappropriate cell proliferation, apoptosis, and genome instability, which in turn promote the likelihood of tumorigenesis. Thus, a comprehensive understanding of the normal regulation of DNA replication as well as mechanisms contributing to its deregulation is critical in the development of novel therapies and diagnostic tools for cancer treatment. The research proposed here will focus on the very first regulatory step in eukaryotic DNA replication, which is the licensing of thousands of replication initiation sites, or origins, throughout the genome. Origins are rendered competent, or licensed, for DNA replication in S phase by the chromatin-loading of a DNA helicase known as the Mini-Chromosome Maintenance Complex (MCM) during G1 phase of the cell cycle. While MCM complexes are constitutively nuclear and can exist in both soluble and chromatin-bound states, only origins containing chromatin-bound MCM complexes are licensed for replication. This licensing step is tightly regulated such that it is ony allowed during G1 phase and prohibited during quiescence (also termed G0), S, G2, and M phases. Molecular mechanisms coordinating these changes in MCM loading status with cell cycle progression remain unclear and are the focus of this proposal. The first aim seeks to determine how unlicensed chromatin is maintained during cellular quiescence. Our lab recently discovered that the stress MAP kinases p38 and JNK inhibit licensing during a cellular stress response, and these kinases are highly active in normal cells during quiescence. Based on these observations, we hypothesize that the stress MAPKs additionally play a role in blocking MCM loading during quiescence to maintain the non-proliferative state. We will test this idea by inhibiting MAPK activity during quiescence and observing effects on licensing competence. The second aim of this proposal addresses the question of how MCM loading in G1 is linked with S phase entry. Given our recent finding that MCM loading is required for initiation of S phase, we postulate that as-yet unidentified proteins interact preferentially with loaded MCM complexes and function to promote the G1/S transition. To discover such mediators of the G1/S transition, we will isolate MCM complexes from soluble and chromatin fractions of G1 cells for comparative mass spectrometry analysis. Proteins interacting exclusively with chromatin-bound MCM complexes in G1 will be the focus of further functional analyses to determine their role in mediating cell cycle progression. Ultimately, we anticipate that the long-term analysis of newly identified origin licensing regulators from these studies will guide the development of novel therapeutics for many human cancers.

描述（由申请人提供）：为了保持适当的基因组完整性，细胞适当协调控制DNA复制的细胞内和细胞外信号通路是至关重要的。对这一基本过程的不当调控最终会导致不适当的细胞增殖、细胞凋亡和基因组不稳定，进而促进肿瘤发生的可能性。因此，全面了解DNA复制的正常调节以及有助于其解除管制的机制对于开发用于癌症治疗的新疗法和诊断工具至关重要。这里提出的研究将集中在真核生物DNA复制的第一个调控步骤，即整个基因组中数千个复制起始位点或起源的许可。在细胞周期的G1期，DNA解旋酶（称为迷你染色体维持复合体（MCM））的染色质装载使起源在S期具有DNA复制能力。虽然MCM复合物是核结构，可以存在于可溶性和染色质结合状态，但只有含有染色质结合的MCM复合物的起源才允许复制。此许可步骤受到严格监管，因此仅在G1阶段允许，在静止（也称为G0）、S、G2和M阶段禁止。在细胞周期进程中协调MCM负载状态变化的分子机制尚不清楚，这也是本研究的重点。第一个目标是确定在细胞静止期间未授权的染色质是如何维持的。我们的实验室最近发现，应激MAP激酶p38和JNK在细胞应激反应中抑制许可，这些激酶在正常细胞处于静止状态时高度活跃。基于这些观察结果，我们假设应激MAPKs在静止期间阻断MCM加载以维持非增殖状态中也起作用。我们将通过在静默期间抑制MAPK活性并观察对许可能力的影响来验证这一观点。本提案的第二个目标解决了G1中MCM加载如何与S相进入相关联的问题。鉴于我们最近的发现，MCM加载是S期起始所必需的，我们假设尚未确定的蛋白质优先与加载的MCM复合物相互作用，并发挥促进G1/S转变的作用。为了发现G1/S过渡的介质，我们将从G1细胞的可溶性和染色质部分分离MCM复合物进行比较质谱分析。G1期仅与染色质结合MCM复合物相互作用的蛋白质将成为进一步功能分析的重点，以确定它们在介导细胞周期进程中的作用。最终，我们预计，对这些研究中新发现的原产地许可监管机构的长期分析将指导许多人类癌症新疗法的开发。