Linking DNA Replication Origin Licensing with Cell Cycle Progression

描述(申请人提供)：为了保持适当的基因组完整性，细胞适当协调控制DNA复制的细胞内和细胞外信号通路是至关重要的。对这一基本过程的不正确调控最终会导致不适当的细胞增殖、凋亡和基因组不稳定，这反过来又会促进肿瘤发生的可能性。因此，全面了解DNA复制的正常调控以及导致其放松调控的机制，对于开发癌症治疗的新疗法和诊断工具至关重要。这里提出的研究将集中在真核DNA复制的第一个调控步骤，即整个基因组中数千个复制起始点或起始点的许可。在细胞周期的G1期，通过一种被称为微染色体维护复合体的DNA解旋酶的染色质负载，起始点被赋予了在S期进行DNA复制的能力或许可。虽然MCM复合体是构成核的，并且可以以可溶状态和染色质结合状态存在，但只有包含染色质结合MCM复合体的起源才被许可进行复制。这一许可步骤受到严格监管，仅在G1阶段允许，在静止(也称为G0)、S、G2和M阶段禁止。协调MCM负荷状态与细胞周期进程的这些变化的分子机制仍不清楚，也是本提案的重点。第一个目标是确定在细胞静止期间，未经许可的染色质是如何维持的。我们的实验室最近发现，应激映射激酶p38和JNK在细胞应激反应中抑制许可，而这些激酶在正常细胞静止时高度活跃。基于这些观察结果，我们推测，应激性MAPK在静止期阻止MCM负荷以维持非增殖状态的另外一个作用。我们将通过在静止时抑制MAPK活性并观察对许可能力的影响来测试这一想法。这项建议的第二个目的是解决G1阶段的MCM加载与S阶段进入如何联系的问题。鉴于我们最近发现MCM负载是启动S相所必需的，我们假设迄今未知的蛋白质优先与负载的MCM复合体相互作用，并促进G1/S的转变。为了发现这种G1/S转变的介体，我们将从G1细胞的可溶性和染色质部分分离出MCM复合体，用于比较质谱分析。与染色质结合的MCM复合体相互作用的蛋白质将是进一步功能分析的重点，以确定它们在调节细胞周期进程中的作用。最终，我们预计对这些研究中新发现的原产地许可监管机构的长期分析将指导许多人类癌症的新疗法的开发。

DESCRIPTION (provided by applicant): To maintain proper genome integrity, it is critical that cells properly coordinate intracellular and extracellular signaling pathways controlling DNA replication. Improper regulation of this fundamental process can ultimately contribute to inappropriate cell proliferation, apoptosis, and genome instability, which in turn promote the likelihood of tumorigenesis. Thus, a comprehensive understanding of the normal regulation of DNA replication as well as mechanisms contributing to its deregulation is critical in the development of novel therapies and diagnostic tools for cancer treatment. The research proposed here will focus on the very first regulatory step in eukaryotic DNA replication, which is the licensing of thousands of replication initiation sites, or origins, throughout the genome. Origins are rendered competent, or licensed, for DNA replication in S phase by the chromatin-loading of a DNA helicase known as the Mini-Chromosome Maintenance Complex (MCM) during G1 phase of the cell cycle. While MCM complexes are constitutively nuclear and can exist in both soluble and chromatin-bound states, only origins containing chromatin-bound MCM complexes are licensed for replication. This licensing step is tightly regulated such that it is ony allowed during G1 phase and prohibited during quiescence (also termed G0), S, G2, and M phases. Molecular mechanisms coordinating these changes in MCM loading status with cell cycle progression remain unclear and are the focus of this proposal. The first aim seeks to determine how unlicensed chromatin is maintained during cellular quiescence. Our lab recently discovered that the stress MAP kinases p38 and JNK inhibit licensing during a cellular stress response, and these kinases are highly active in normal cells during quiescence. Based on these observations, we hypothesize that the stress MAPKs additionally play a role in blocking MCM loading during quiescence to maintain the non-proliferative state. We will test this idea by inhibiting MAPK activity during quiescence and observing effects on licensing competence. The second aim of this proposal addresses the question of how MCM loading in G1 is linked with S phase entry. Given our recent finding that MCM loading is required for initiation of S phase, we postulate that as-yet unidentified proteins interact preferentially with loaded MCM complexes and function to promote the G1/S transition. To discover such mediators of the G1/S transition, we will isolate MCM complexes from soluble and chromatin fractions of G1 cells for comparative mass spectrometry analysis. Proteins interacting exclusively with chromatin-bound MCM complexes in G1 will be the focus of further functional analyses to determine their role in mediating cell cycle progression. Ultimately, we anticipate that the long-term analysis of newly identified origin licensing regulators from these studies will guide the development of novel therapeutics for many human cancers.