Cell Cycle Regulation of Vertebrate DNA Replication

描述(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复制一次DNA。在G1期，复制前复合体(Pre-RCs)通过ORC、Cdc6、Cdt1和MCM2-7的顺序招募在起点组装。在S阶段，当DNA复制开始时，前RC被拆解，严格禁止新的前RC组装。因此，每个起始点只发生一个启动事件，DNA复制被限制在一轮。在脊椎动物中，由于双黄素和泛素介导的CDT1蛋白分解，Pre-RC组装被阻止在S期。利用从非洲爪哇卵提取液中提取的无细胞系统，该实验室获得了令人惊讶的发现：在S期，CDT1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复制分叉处的过程性因子PCNA相互作用。来自其他实验室的研究表明，依赖于增殖细胞核抗原的CDT1破坏在人类、苍蝇、蠕虫和可能的裂变酵母中是保守的。这条途径代表了一种新的模式，在这种模式中，蛋白质降解的时间控制涉及到底物(CDT1)的激活，它通过对接到细胞周期调节结构(染色质结合的增殖细胞核抗原)上。这一提议包含了一些实验，这些实验将阐明依赖于增殖细胞核抗原的CDT1破坏受到时间控制的机制。在特定的目标1中，我们讨论了CDT1如何选择性地与染色质结合的增殖细胞核抗原相互作用，以确保CDT1通常在G1中永远不会被破坏。在特定的目标2中，我们表征了CDT1的退化基序，并研究了它如何介导与增殖细胞核抗原和CUL4-DDB1CDt2的结合。在特定的目标3中，我们在一个纯化的系统中重建了CDT1泛素化，以便我们可以区分不同的机制，即增殖细胞核抗原如何刺激这一过程。最后，在特定的目标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，并测试了一个通过增殖细胞核抗原/CUL4-DDB1CDt2通路来局部控制蛋白降解的新模型。总之，这些研究将阐明维持基因组稳定的一种新的蛋白质分解途径背后的分子机制。为了保持我们基因组的完整性并预防癌症等疾病，细胞在每次细胞分裂之前制作一份精确的DNA拷贝是至关重要的。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，它涉及在第一轮染色体复制开始后破坏一种名为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与人类癌症有关，并已被证明会在老鼠身上引起癌症。

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.