Replication licensing and cell cycle checkpoints

描述(由申请人提供)：精确复制整个人类基因组需要数千个起始点获得许可，然后每个细胞周期恰好启动一次复制。起源许可是通过在细胞周期的G1期的起始处组装复制前复合体来完成的。许可太少的源会导致复制不完整，而许可已复制的源会导致重新复制。这两种情况都是基因组不稳定的来源，可能会促进细胞的异常增殖和肿瘤发生。细胞复制的启动是由细胞周期蛋白依赖性蛋白激酶(CDK)的活性刺激的，CDK在G1/S相变时被激活，并一直保持活跃直到有丝分裂。为了防止任何序列被多次复制，在S阶段，一旦开始复制，就禁止起始点许可。在G1之后抑制起始点许可以避免再次复制的机制包括来自刺激复制起始的相同的周期蛋白依赖蛋白激酶(CDK)的关键贡献。这些CDK的功能是阻止复制前复合体在G1后的组装。CDK在复制控制中的双重功能的结果是，在S期开始之前，必须在G1期(CDK活性低的时期)完全完成起源许可，以便整个基因组能够复制，但目前尚不清楚细胞如何确保在S期开始之前完成起源许可。CDK在防止不适当地重新许可起源方面的作用的第二个后果是，许多环境变化触发了细胞周期检查点，通过抑制CDK来阻止细胞周期。如果在S阶段开始后遇到这样的变化--例如DNA损伤或细胞压力--那么一旦细胞从检查站停滞中恢复过来，就有可能重新复制。为了避免再复制及其相关的基因组不稳定，这种细胞周期检查点还必须通过独立于CDK活性的机制来抑制起源许可。此建议解决了复制许可控制和各个检查点响应之间的三个不同接口。其目的是1)确定起源许可与CDK激活和S阶段进入之间的联系，2)确定在DNA损伤后从起源释放许可蛋白CDC6的机制，以及3)确定在细胞应激反应中阻止起源许可的机制。我们的方法依赖于对培养的人类细胞中特定蛋白质和活动的操纵。我们评估了这些操作对基本复制和检查点蛋白的丰度、活性和定位的影响，并确定了这些操作对蛋白质-蛋白质和蛋白质-DNA相互作用的影响。我们预计，全面了解原产地许可规则将有助于人类癌症和其他涉及细胞异常增殖的疾病的诊断、分类和治疗。公共卫生相关性虽然很明显癌细胞不适当地增殖，但仍不完全了解这些细胞与正常细胞的不同之处，或者是什么控制了它们向转移性疾病的发展。DNA复制调控中的错误是癌症发展的主要原因，这一提议旨在获得对DNA复制第一步的调控的新见解，即将必要的复制蛋白定位到DNA。具体的重点是这一初始步骤与细胞对细胞内或细胞外环境扰动的反应的协调。

DESCRIPTION (provided by applicant): Precise duplication of the entire human genome requires that thousands of origins are licensed and then initiate replication exactly once per cell cycle. Origin licensing is accomplished through the assembly of prereplication complexes at origins during the G1 phase of the cell cycle. Licensing too few origins results in incomplete replication, and licensing origins that have already been replicated results in rereplication. Both situations are sources of genome instability that can promote aberrant cell proliferation and oncogenesis. Replication initiation is stimulated by the activity of cyclin dependent protein kinases (Cdks) that are activated at the G1/S phase transition and remain active until mitosis. To prevent any sequence from being replicated more than once, origin licensing is inhibited once replication initiation begins in S phase. The mechanisms that inhibit origin licensing after G1 to avoid rereplication include a critical contribution from the same cyclin dependent protein kinases (Cdks) that stimulate replication initiation. These Cdks function to block the assembly of prereplication complexes after G1. A consequence of the dual function of Cdks in replication control is that origin licensing must be fully completed in G1 (during the period of low Cdk activity) before S phase begins so that the entire genome can be replicated, but it is not known how cells ensure that origin licensing is complete before S phase begins. A second consequence of the role of Cdks in preventing inappropriate re-licensing of origins is that many environmental changes trigger cell cycle checkpoints that arrest the cell cycle by inhibiting Cdks. If such changes - such as DNA damage or cellular stress - are encountered after S phase begins, then cells risk rereplication once they recover from the checkpoint arrest. To avoid rereplication and its associated genome instability, such cell cycle checkpoints must also inhibit origin licensing by mechanisms that are independent of Cdk activity. This proposal addresses three distinct interfaces between replication licensing control and individual checkpoint responses. The aims are to 1) Determine the mechanism that links origin licensing to Cdk activation and S phase entry, 2) Determine the mechanism controlling release of the licensing protein, Cdc6, from origins after DNA damage, and 3) Determine the mechanism that prevents origin licensing during a cellular stress response. Our methods rely on the manipulation of specific proteins and activities in cultured human cells. We evaluate the effects of these manipulations on the abundance, activity and localization of essential replication and checkpoint proteins, and we determine the effects of these manipulations on protein- protein and protein-DNA interactions. We anticipate that a comprehensive understanding of the regulation of origin licensing will facilitate the diagnosis, classification, and treatment of human cancers as well as other diseases that involve aberrant cell proliferation. PUBLIC HEALTH RELEVANCE Although it is clear that cancer cells proliferate inappropriately, it is still not fully understood how these cells differ from normal cells or what controls their progression to metastatic disease. Errors in the regulation of DNA replication are major contributors to cancer development, and this proposal is designed to gain new insight into the regulation of the first step in DNA replication, the localization of essential replication proteins to DNA. The specific focus is on the coordination of this initial step with the cellular responses to perturbations in the intracellular or extracellular environment.