Cell Cycle Regulation of Vertebrate DNA Replication

脊椎动物 DNA 复制的细胞周期调控

• 批准号: 7498477
• 负责人: Johannes Walter
• 金额: $ 32.13万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7498477
• 关键词: Address; Binding; Cancer Biology; Cancer Etiology; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell division; Cell-Free System; Cells; Chromatin; Chromatin Structure; Chromosomes; Complex; Coupled; DNA; DNA Binding; DNA Replication Factor; DNA biosynthesis; DNA replication fork; DNA replication origin; DNA-Directed DNA Polymerase; Discrimination; Disease; Docking; Eukaryotic Cell; Event; Failure; Figs - dietary; Fission Yeast; Fluorescence Microscopy; G1 Phase; Geminin; Genome; Genome Stability; Genomic Instability; Genomics; Health; Human; Imaging Techniques; Individual; Laboratories; Left; Licensing; Malignant Neoplasms; Measures; Mediating; Modeling; Molecular; Mus; Organism; PCNA gene; Pathway interactions; Phase; Phosphorylation; Pre-Replication Complex; Process; Prohibit; Property; Protein Kinase; Proteins; Proteolysis; Reaction; Reliance; Replication Initiation; Replication Licensing; Replication Origin; Role; System; Testing; Time; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Vertebrates; Work; Xenopus; egg; fly; novel; prevent; reconstitution; research study; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): To maintain genomic integrity, eukaryotic cells duplicate their DNA precisely once before each cell division. In the G1 phase, pre-replication complexes (pre-RCs) are assembled at origins via the sequential recruitment of ORC, Cdc6, Cdt1, and MCM2-7. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is strictly prohibited. As a result, only one initiation event occurs at each origin, and DNA replication is limited to a single round. In vertebrates, pre-RC assembly is blocked in S phase due to Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, this laboratory made the surprising discovery that the destruction of Cdt1 in S phase is intimately coupled to DNA replication. Cdt1 is ubiquitylated on chromatin by the E3 ubiquitin ligase Cul4-Ddb1Cdt2, and this ubiquitylation event requires the interaction of Cdt1 with the processivity factor PCNA at the DNA replication fork. Studies from other laboratories suggest that PCNA-dependent Cdt1 destruction is conserved in humans, flies, worms, and possibly fission yeast. This pathway represents a new paradigm in which temporal control of proteolysis involves activation of a substrate (Cdt1) via its docking onto a cell-cycle regulated structure (chromatin-bound PCNA). This proposal contains experiments that will elucidate the mechanism by which PCNA-dependent Cdt1 destruction is temporally controlled. In Specific Aim 1, we address how Cdt1 interacts selectively with chromatin-bound PCNA to insure that Cdt1 is normally never destroyed in G1. In Specific Aim 2, we characterize the degron motif of Cdt1 and examine how it mediates binding to PCNA and Cul4-Ddb1Cdt2. In Specific Aim 3, we reconstitute Cdt1 ubiquitylation in a purified system so that we may distinguish between different mechanisms for how PCNA stimulates this process. Finally, in Specific Aim 4, we use real-time fluorescence microscopy to address how rapidly Cdt1 is destroyed at S phase onset, and we test a new model for local control of proteolysis by the PCNA/Cul4-Ddb1Cdt2 pathway. Together, these studies will illuminate the molecular mechanism underlying a novel proteolysis pathway that maintains genome stability. Narrative To maintain the integrity of our genomes and prevent diseases such as cancer, it is crucial that cells make one precise copy of their DNA before each cell division. This laboratory has discovered a new mechanism, which insures the accuracy of genome duplication in all higher organisms, and it involves the destruction of a DNA replication factor called Cdt1 after the first round of chromosome duplication has been initiated. The work is highly relevant for human health because failure to destroy Cdt1 is correlated with human cancer, and has been shown to cause cancer in mice.

描述（由申请人提供）：为了保持基因组完整性，真核细胞在每次细胞分裂前精确地复制其 DNA 一次。在 G1 期，复制前复合物 (pre-RC) 通过 ORC、Cdc6、Cdt1 和 MCM2-7 的顺序募集在起始点组装。在S期，当DNA复制开始时，前RC被分解，并且严格禁止新的前RC组装。因此，每个起点仅发生一个起始事件，DNA 复制仅限于一轮。在脊椎动物中，由于 Geminin 和泛素介导的 Cdt1 蛋白水解作用，前 RC 组装在 S 期被阻断。该实验室使用源自非洲爪蟾卵提取物的无细胞系统，得出了令人惊讶的发现：S 期 Cdt1 的破坏与 DNA 复制密切相关。 Cdt1 通过 E3 泛素连接酶 Cul4-Ddb1Cdt2 在染色质上泛素化，这种泛素化事件需要 Cdt1 与 DNA 复制叉处的持续因子 PCNA 相互作用。其他实验室的研究表明，PCNA 依赖性 Cdt1 破​​坏在人类、果蝇、蠕虫和可能的裂殖酵母中是保守的。该途径代表了一种新的范例，其中蛋白水解的时间控制涉及通过将底物 (Cdt1) 对接到细胞周期调节结构（染色质结合的 PCNA）上来激活底物 (Cdt1)。该提案包含的实验将阐明 PCNA 依赖性 Cdt1 破​​坏被暂时控制的机制。在具体目标 1 中，我们讨论了 Cdt1 如何选择性地与染色质结合的 PCNA 相互作用，以确保 Cdt1 在 G1 中通常不会被破坏。在具体目标 2 中，我们描述了 Cdt1 的降解决定子基序，并研究了它如何介导与 PCNA 和 Cul4-Ddb1Cdt2 的结合。在具体目标 3 中，我们在纯化系统中重建了 Cdt1 泛素化，以便我们可以区分 PCNA 如何刺激这一过程的不同机制。最后，在具体目标 4 中，我们使用实时荧光显微镜来研究 Cdt1 在 S 期开始时被破坏的速度，并测试了 PCNA/Cul4-Ddb1Cdt2 途径局部控制蛋白水解的新模型。这些研究将共同​​阐明维持基因组稳定性的新型蛋白水解途径的分子机制。 叙述 为了保持基因组的完整性并预防癌症等疾病，细胞在每次细胞分裂前精确复制 DNA 至关重要。该实验室发现了一种新机制，可确保所有高等生物体基因组复制的准确性，其中涉及在第一轮染色体复制开始后破坏称为 Cdt1 的 DNA 复制因子。这项工作与人类健康高度相关，因为未能破坏 Cdt1 与人类癌症相关，并且已被证明会导致小鼠癌症。