Post-translational regulation of DNA replication origin licensing in human cells

人类细胞 DNA 复制起点许可的翻译后调控

• 批准号: 10093060
• 负责人: Jeanette Gowen Cook
• 金额: $ 41.81万
• 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: 2013-08-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093060
• 关键词: Abnormal Cell; Address; Aging; Binding; Biochemistry; Bypass; CCNE1 gene; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cells; Chronic; Complex; Custom; Cyclin A; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Cytoplasm; DNA; DNA biosynthesis; DNA replication origin; Data; Defect; Degenerative Disorder; Development; Disease; Enzymes; Epithelial Cells; Failure; Flow Cytometry; Funding; Future; G1 Phase; G1/S Transition; G2 Phase; Genome Stability; Genomic Instability; Goals; Human; Human Genome; Hypersensitivity; Image Cytometry; Impairment; Individual; Intuition; Knowledge; Lead; Licensing; Light; Malignant Neoplasms; Mammalian Cell; Mammals; Measurement; Mediating; Methods; Mitosis; Molecular; Molecular Biology; Mutagenesis; Pathologic; Pathway interactions; Pattern; Phase Transition; Phosphorylation; Phosphotransferases; Post-Translational Regulation; Precision therapeutics; Protein Dephosphorylation; Protein Kinase; Proteins; Regulation; Replication Origin; Research; Role; S Phase; Seminal; Series; Signal Pathway; System; Testing; Thinking; Yeasts; cyclin G1; design; extracellular; genotoxicity; helicase; human disease; innovation; insight; live cell imaging; personalized diagnostics; preservation; prevent; regenerative therapy; replication stress; single cell analysis; success; tumorigenesis; ubiquitin-protein ligase

PHOSPHOREGULATION OF DNA REPLICATION ORIGIN LICENSING IN MAMMALIAN CELLS This proposal seeks new insight into the fundamental organization of the human cell division cycle and how perturbations to that organization lead to genome instability and pathological states. Complete and efficient duplication of the entire human genome requires that many thousands of DNA replication origins become “licensed” in G1 of each cell division cycle through the loading of MCM helicase complexes. DNA-loaded MCM complexes are then activated during S phase. Loss of normal origin licensing control causes hypersensitivity to replication stress and induces genome instability, which can ultimately lead to oncogenesis, developmental defects, and degeneration. Our long-term goal is to understand how DNA replication origin licensing control is coordinated with intracellular and extracellular signaling pathways that control proliferation and development. We hypothesize that perturbations to this coordination cause genome instability and proliferation failure. Our experimental approach is a combination of quantitative single cell analyses with molecular biology and biochemistry using cultured human cells. We focus on uncovering molecular mechanisms and dynamics, and then testing the cellular consequences of disrupting those mechanisms. Our recent progress, innovative experimental strategies, and preliminary data inspire a new series of projects to address these specific goals: 1) determine precisely how changes in individual cyclin/CDK enzymes impact origin licensing and the G1 to S phase transition 2) define the molecular consequences phosphorylating an essential origin licensing protein, Cdt1, and 3) define the relationships among CDK-mediated phosphorylation, the APC/C E3 ubiquitin ligase, and origin licensing, particularly for regulation of the Cdc6 licensing protein and preventing re-replication. Success will shed light on the mechanisms that can drive mutagenesis, cancer, cell death, and aging. The deep understanding of cell proliferation control sought through the pursuit of these aims will have downstream implications for efforts to precisely define and treat human disease and for future regenerative therapies.

哺乳动物细胞DNA复制起始许可的磷酸化调节 这项提案旨在对人类细胞分裂周期的基本组织以及如何进行细胞分裂的新见解。 对该组织的扰动导致基因组不稳定和病理状态。完整高效 复制整个人类基因组需要成千上万的DNA复制起点， 在每个细胞分裂周期的G1期通过加载MCM解旋酶复合物而“许可”。DNA负载MCM 然后在S期期间激活复合物。正常原产地许可控制的丧失导致对 复制应激并诱导基因组不稳定性，这最终可导致肿瘤发生、发育 缺陷和退化。我们的长期目标是了解DNA复制起点许可控制是如何 与控制增殖和发育的细胞内和细胞外信号通路协调。 我们假设，这种协调的扰动导致基因组不稳定和增殖失败。我们 实验方法是定量单细胞分析与分子生物学的结合， 使用培养的人类细胞进行生物化学。我们专注于揭示分子机制和动力学， 然后测试破坏这些机制的细胞后果。我们最近的进展，创新 实验策略和初步数据激发了一系列新的项目，以解决这些具体目标：1） 精确确定单个细胞周期蛋白/CDK酶的变化如何影响来源许可和G1到S 相变2）定义磷酸化必需来源许可蛋白的分子结果， Cdt 1，和3）定义CDK介导的磷酸化，APC/CE 3泛素连接酶，和 原产地许可，特别是用于调节Cdc 6许可蛋白和防止再复制。成功 将揭示可以驱动诱变，癌症，细胞死亡和衰老的机制。深 通过追求这些目标而寻求的对细胞增殖控制的理解将具有下游 对精确定义和治疗人类疾病以及未来再生疗法的影响。