Cell Cycle Regulation of Vertebrate DNA Replication

描述（由申请人提供）：为了保持基因组的完整性，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的顺序募集在起点组装。在S期，当DNA复制开始时，pre-RC被分解，并且严格禁止新的pre-RC组装。因此，在每个起始点只发生一次起始事件，DNA复制被限制在单轮。在脊椎动物中，由于gemini和泛素介导的Cdt1蛋白水解，pre-RC组装在S期被阻断。利用从爪蟾卵提取物中提取的无细胞系统，该实验室令人惊讶地发现，Cdt1在S期的破坏与DNA复制密切相关。Cdt1在染色质上被E3泛素连接酶Cul4-Ddb1Cdt2泛素化，这种泛素化事件需要Cdt1与DNA复制叉上的加工因子PCNA相互作用。来自其他实验室的研究表明，依赖于pcna的Cdt1破坏在人类、苍蝇、蠕虫和可能的裂变酵母中是保守的。这一途径代表了一种新的模式，其中蛋白质水解的时间控制涉及通过对接到细胞周期调节结构（染色质结合的PCNA）的底物（Cdt1）的激活。这一建议包含的实验将阐明的机制，由pcna依赖的Cdt1破坏是暂时控制。在Specific Aim 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Specific Aim 2中，我们描述了Cdt1的退化基序，并研究了它如何介导与PCNA和Cul4-Ddb1Cdt2的结合。在特异性目标3中，我们在纯化系统中重建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Specific Aim 4中，我们使用实时荧光显微镜来解决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。总之，这些研究将阐明维持基因组稳定性的一种新的蛋白质水解途径的分子机制。为了保持我们基因组的完整性并预防癌症等疾病，细胞在每次分裂前精确复制一份DNA是至关重要的。这个实验室发现了一种新的机制，它确保了所有高等生物基因组复制的准确性，它涉及到在第一轮染色体复制开始后，一个名为Cdt1的DNA复制因子的破坏。这项工作与人类健康高度相关，因为破坏Cdt1的失败与人类癌症有关，并且已被证明会导致小鼠癌症。

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.