Origin Recognition Complex and Transcriptional Silencers

描述（由申请人提供）：在真核细胞中，起点识别复合物（ORC）直接结合并从而选择DNA复制起点，即染色体上DNA复制起始的位点。虽然ORC起源的相互作用是真核基因组的复制和稳定性的基础，但调节这些相互作用的分子机制知之甚少。我们在酿酒酵母的最后一个资助周期中的发现使我们处于有利地位，以解决与ORC在DNA复制起始中的作用相关的两个关键问题。首先，我们发现，已建立的酵母ORC-DNA接口是不够的，以解释ORC结合到大部分的起源和ORC-染色质相互作用的贡献更显着的起源选择芽殖酵母比以前认识到。因此，提出的后生动物，一个ORC-染色质界面是重要的DNA复制起点的选择，在芽殖酵母中的ORC。我们已经确定了“染色质依赖性”酵母的起源，这将使我们能够使用强大的实验工具，酵母定义的ORC染色质接口的分子细节，从而提供新的见解ORC如何选择染色体内的DNA复制起点。染色质、其调节剂和DNA复制蛋白之间在真核生物中的强保守性意味着我们在芽殖酵母中定义的ORC-染色质界面的实质性方面将与人类细胞中的类似界面相关和/或可用于操纵该界面以改善人类健康。其次，越来越多的证据表明，ORC与染色体和起源的关系比通常描述或理解的更动态。此外，数据表明，这些动力学相关的起源在体内的调节。这些数据和我们自己最近的观察集中在“染色质依赖性”起源的功能，导致我们以下的假设：ORC-起源动力学的差异，这是由ORC-染色质和ORC-DNA相互作用在个别起源的差异，有助于调节起源激活的差异，存在于100到1000的起源，用于复制真核基因组。我们将测试这一假设，从而获得新的和重要的见解基本的，但相对未探索的问题，如何ORC的起源相互作用调节起源激活体内。

DESCRIPTION (provided by applicant): In eukaryotic cells, the origin recognition complex (ORC) binds directly to and thereby selects DNA replication origins, the sites on chromosomes where DNA replication initiates. While ORC-origin interactions are fundamental to the duplication and stability of the eukaryotic genome, the molecular mechanisms that regulate these interactions are poorly understood. Our findings in the last grant cycle in Saccharomyces cerevisiae place us in a strong position to address two critical issues relevant to ORC's role in DNA replication initiation. First, we discovered that the established yeast ORC-DNA interface is insufficient to explain ORC binding to a large fraction of origins and that ORC-chromatin interactions contribute more substantially to origin selection in budding yeast than previously appreciated. Thus as proposed for metazoans, an ORC-chromatin interface is important for the selection of DNA replication origins by ORC in budding yeast. We have identified 'chromatin-dependent' yeast origins that will allow us to use the powerful experimental tools of yeast to define the ORC-chromatin interface in molecular detail and thus provide new insights into how ORC selects DNA replication origins within chromosomes. The strong conservation between chromatin, its regulators and the DNA replication proteins across eukaryotes means that substantial aspects of the ORC-chromatin interface we define in budding yeast will be relevant to the analogous interface in human cells and/or useful for manipulating this interface for improving human health. Second, increasing lines of evidence from a variety of systems indicate that ORC has a more dynamic relationship with chromosomes and origins than commonly depicted or appreciated. Moreover, data suggest that these dynamics are relevant to origin regulation in vivo. These data and our own recent observations focused on the function of 'chromatin-dependent' origins lead us to the following hypothesis: Differences in ORC-origin dynamics, which are caused by differences in ORC-chromatin and ORC-DNA interactions at individual origins, contribute to regulating differences in origin activation that exist among the 100's to 1000's of origins that are used to replicate a eukaryotic genome. We will test this hypothesis and thereby gain new and important insights into the basic but relatively unexplored issue of how ORC-origin interactions regulate origin activation in vivo.