Diversity Supplement: Post-Translational Regulation of DNA Replication Origin Licensing in Human Cells

这一提议旨在对人类细胞分裂周期的基本组织进行新的了解 以及对该组织的扰动如何导致基因组不稳定和病理状态。 完整而有效地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期通过装载 MCM解旋酶复合物。然后在S期期间活化DNA负载的MCM复合物。损失 正常来源许可控制引起对复制应激的超敏反应，并诱导基因组 不稳定性，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如何协调DNA复制起点许可控制 细胞内和细胞外信号通路控制增殖和发育。 我们假设，这种协调的扰动会导致基因组的不稳定和增殖 失败我们的实验方法是定量单细胞分析与 分子生物学和生物化学。我们专注于发现 分子机制和动力学，然后测试破坏细胞的后果， 这些机制。我们最近的进展，创新的实验策略和初步数据 激发了一系列新的项目来实现这些具体目标：1）准确地确定如何 单个细胞周期蛋白/CDK酶的变化影响起源许可和G1到S期的转变 2)定义磷酸化必需来源许可蛋白Cdt 1的分子结果， 以及3）确定CDK介导的磷酸化、APC/CE 3泛素化和细胞凋亡之间的关系。 连接酶和来源许可，特别是用于调节Cdc 6许可蛋白和防止 再复制这一成功将有助于揭示基因突变、癌症、细胞分裂和细胞凋亡的机制。 死亡和衰老通过追求对细胞增殖控制的深入理解 这些目标将对精确定义和治疗人类疾病的努力产生下游影响。 疾病和未来的再生疗法。

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.