Replication licensing and cell cycle checkpoints

复制许可和细胞周期检查点

• 批准号: 8019565
• 负责人: Jeanette Gowen Cook
• 金额: $ 26.46万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019565
• 关键词: Address; Apoptosis; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Proliferation; Cells; Cellular Stress; Cellular Stress Response; Checkpoint kinase 1; Chromatin; Classification; Complex; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA biosynthesis; DNA damage checkpoint; DNA-Protein Interaction; Defect; Development; Diagnosis; Disease; Ensure; Environment; G1 Arrest; G1 Phase; Genome; Genome Stability; Genomic Instability; Health; Human; Human Genome; Individual; Investigation; Licensing; Licensing Factor; Link; MAPK8 gene; Malignant Neoplasms; Metastatic to; Methods; Mitogen-Activated Protein Kinases; Mitosis; Modeling; Normal Cell; Pathway interactions; Phase Transition; Phosphorylation; Proliferating; Protein Kinase; Protein p53; Proteins; Regulation; Replication Initiation; Replication Licensing; Risk; Role; S Phase; Signal Transduction; Source; Stress; Testing; cancer cell; controlled release; cyclin-dependent kinase-activating kinase; design; environmental change; extracellular; insight; mitogen-activated protein kinase p38; novel; origin recognition complex; prevent; protein protein interaction; response; tumorigenesis

DESCRIPTION (provided by applicant): Precise duplication of the entire human genome requires that thousands of origins are licensed and then initiate replication exactly once per cell cycle. Origin licensing is accomplished through the assembly of prereplication complexes at origins during the G1 phase of the cell cycle. Licensing too few origins results in incomplete replication, and licensing origins that have already been replicated results in rereplication. Both situations are sources of genome instability that can promote aberrant cell proliferation and oncogenesis. Replication initiation is stimulated by the activity of cyclin dependent protein kinases (Cdks) that are activated at the G1/S phase transition and remain active until mitosis. To prevent any sequence from being replicated more than once, origin licensing is inhibited once replication initiation begins in S phase. The mechanisms that inhibit origin licensing after G1 to avoid rereplication include a critical contribution from the same cyclin dependent protein kinases (Cdks) that stimulate replication initiation. These Cdks function to block the assembly of prereplication complexes after G1. A consequence of the dual function of Cdks in replication control is that origin licensing must be fully completed in G1 (during the period of low Cdk activity) before S phase begins so that the entire genome can be replicated, but it is not known how cells ensure that origin licensing is complete before S phase begins. A second consequence of the role of Cdks in preventing inappropriate re-licensing of origins is that many environmental changes trigger cell cycle checkpoints that arrest the cell cycle by inhibiting Cdks. If such changes - such as DNA damage or cellular stress - are encountered after S phase begins, then cells risk rereplication once they recover from the checkpoint arrest. To avoid rereplication and its associated genome instability, such cell cycle checkpoints must also inhibit origin licensing by mechanisms that are independent of Cdk activity. This proposal addresses three distinct interfaces between replication licensing control and individual checkpoint responses. The aims are to 1) Determine the mechanism that links origin licensing to Cdk activation and S phase entry, 2) Determine the mechanism controlling release of the licensing protein, Cdc6, from origins after DNA damage, and 3) Determine the mechanism that prevents origin licensing during a cellular stress response. Our methods rely on the manipulation of specific proteins and activities in cultured human cells. We evaluate the effects of these manipulations on the abundance, activity and localization of essential replication and checkpoint proteins, and we determine the effects of these manipulations on protein- protein and protein-DNA interactions. We anticipate that a comprehensive understanding of the regulation of origin licensing will facilitate the diagnosis, classification, and treatment of human cancers as well as other diseases that involve aberrant cell proliferation. PUBLIC HEALTH RELEVANCE Although it is clear that cancer cells proliferate inappropriately, it is still not fully understood how these cells differ from normal cells or what controls their progression to metastatic disease. Errors in the regulation of DNA replication are major contributors to cancer development, and this proposal is designed to gain new insight into the regulation of the first step in DNA replication, the localization of essential replication proteins to DNA. The specific focus is on the coordination of this initial step with the cellular responses to perturbations in the intracellular or extracellular environment.

描述（由申请人提供）：整个人类基因组的精确复制需要数千个起始点获得许可，然后在每个细胞周期恰好启动一次复制。起源许可是通过在细胞周期的G1期在起源处组装复制前复合物来完成的。授权的起始点太少会导致不完整的复制，而授权已复制的起始点会导致重新复制。这两种情况都是基因组不稳定性的来源，可以促进异常细胞增殖和肿瘤发生。细胞周期蛋白依赖性蛋白激酶（Cdks）的活性刺激复制起始，Cdks在G1/S期转变时被激活并保持活性直至有丝分裂。为了防止任何序列被复制一次以上，一旦在S期开始复制启动，就禁止原始许可。抑制G1后的起源许可以避免再复制的机制包括来自刺激复制起始的相同细胞周期蛋白依赖性蛋白激酶（Cdks）的关键贡献。这些Cdks的功能是阻止G1后复制前复合物的组装。Cdk在复制控制中的双重功能的一个后果是，起源许可必须在S期开始之前的G1期（在低Cdk活性期间）完全完成，以便整个基因组可以复制，但目前尚不清楚细胞如何确保S期开始之前完成起源许可。Cdks在防止来源的不适当再许可中的作用的第二个后果是，许多环境变化触发细胞周期检查点，其通过抑制Cdks来阻止细胞周期。如果在S期开始后遇到这样的变化，如DNA损伤或细胞应激，那么一旦细胞从检查点停滞中恢复，它们就有重新复制的风险。为了避免再复制及其相关的基因组不稳定性，这样的细胞周期检查点还必须通过独立于Cdk活性的机制抑制起源许可。此建议解决了复制许可控制和各个检查点响应之间的三个不同接口。目的是1）确定将来源许可与Cdk激活和S期进入联系起来的机制，2）确定控制许可蛋白Cdc 6在DNA损伤后从来源释放的机制，以及3）确定在细胞应激反应期间阻止来源许可的机制。我们的方法依赖于对培养的人类细胞中特定蛋白质和活性的操作。我们评估了这些操作对必需复制和检查点蛋白的丰度、活性和定位的影响，并确定了这些操作对蛋白质-蛋白质和蛋白质-DNA相互作用的影响。我们预计，对原产地许可监管的全面了解将有助于人类癌症以及其他涉及异常细胞增殖的疾病的诊断、分类和治疗。 虽然很明显癌细胞会不适当地增殖，但仍然没有完全理解这些细胞与正常细胞的区别，或者是什么控制了它们向转移性疾病的进展。DNA复制调控中的错误是癌症发展的主要贡献者，该提案旨在获得对DNA复制第一步调控的新见解，即将必需的复制蛋白定位于DNA。具体的重点是协调这一初始步骤与细胞内或细胞外环境中的扰动的细胞反应。