Replication licensing and cell cycle checkpoints

描述（由申请人提供）：整个人类基因组的精确复制需要数千个起源获得许可，然后每个细胞周期精确地启动一次复制。起源许可是通过细胞周期G1期起源处复制前复合体的组装完成的。许可过少的起源会导致复制不完整，而许可已经被复制的起源会导致复制不完整。这两种情况都是基因组不稳定的来源，可以促进异常细胞增殖和肿瘤发生。细胞周期蛋白依赖蛋白激酶（Cdks）在G1/S期被激活，并保持活性直到有丝分裂。为了防止任何序列被复制超过一次，一旦复制开始于S期，起源许可就被抑制。G1后抑制起源许可以避免重复的机制包括刺激复制起始的周期蛋白依赖性蛋白激酶（Cdks）的关键贡献。这些Cdks的作用是阻断G1后复制前复合体的组装。Cdks在复制控制中的双重功能的结果是，在S期开始之前，起始许可必须在G1（在低Cdk活性期间）完全完成，以便整个基因组可以复制，但细胞如何确保起始许可在S期开始之前完成尚不清楚。Cdks在防止不适当的起源再许可中的作用的第二个后果是，许多环境变化触发细胞周期检查点，通过抑制Cdks来阻止细胞周期。如果这种变化——比如DNA损伤或细胞压力——在S期开始后发生，那么一旦细胞从检查点阻滞中恢复过来，它们就会面临风险。为了避免rereplication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活性的机制抑制起源许可。该建议解决了复制许可控制和单个检查点响应之间的三个不同接口。目的是：1)确定将起始许可与Cdk激活和S期进入联系起来的机制；2)确定DNA损伤后起始许可蛋白Cdc6释放的控制机制；3)确定细胞应激反应中阻止起始许可的机制。我们的方法依赖于在培养的人类细胞中操纵特定的蛋白质和活性。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，对原产地许可监管的全面了解将有助于人类癌症以及涉及异常细胞增殖的其他疾病的诊断、分类和治疗。虽然癌细胞的不适当增殖是很清楚的，但人们仍然不完全了解这些细胞与正常细胞有何不同，或者是什么控制了它们向转移性疾病的进展。DNA复制调控中的错误是癌症发展的主要原因，本研究旨在对DNA复制的第一步调控，即基本复制蛋白在DNA上的定位有新的认识。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.