Linking DNA Replication Origin Licensing with Cell Cycle Progression

描述（由申请人提供）：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传导途径至关重要。对这一基本过程的不当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复制的正常调节以及导致其失调的机制对于开发用于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位点或起点的许可。在细胞周期的G1期，通过DNA解旋酶的染色质加载，即微型染色体维持复合物（MCM），使起源能够或被许可在S期进行DNA复制。虽然MCM复合物是组成性核，可以存在于可溶性和染色质结合的状态，只有含有染色质结合MCM复合物的起源被许可复制。该许可步骤受到严格管制，使得其仅在G1阶段期间被允许，并且在静止（也称为G 0）、S、G2和M阶段期间被禁止。协调MCM负载状态与细胞周期进展的这些变化的分子机制仍不清楚，是本提案的重点。第一个目的是确定如何未经许可的染色质是维持在细胞静止。我们的实验室最近发现，应激MAP激酶p38和JNK抑制细胞应激反应过程中的许可，这些激酶在正常细胞在静止期高度活跃。基于这些观察结果，我们假设应激MAPK在静止期阻断MCM负载以维持非增殖状态中另外起作用。我们将测试这个想法，通过抑制MAPK活性在静止和观察许可能力的影响。该建议的第二个目的解决了G1中MCM加载如何与S相进入相关联的问题。鉴于我们最近的发现，MCM加载所需的S相的启动，我们假设，尚未确定的蛋白质相互作用优先与加载MCM复合物和功能，以促进G1/S过渡。为了发现这样的介质的G1/S转换，我们将分离MCM复合物的可溶性和染色质组分的G1细胞进行比较质谱分析。专门与染色质结合的MCM复合物在G1期的蛋白质相互作用将是进一步的功能分析，以确定其在介导细胞周期进程中的作用的焦点。最终，我们预计，从这些研究中新确定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.