Origin Recognition Complex and Transcriptional Silencers

描述(申请人提供)：在真核细胞中，起源识别复合体(ORC)直接与DNA复制起点结合，从而选择DNA复制起点，DNA复制起点位于染色体上。虽然ORC-起源的相互作用是真核基因组复制和稳定的基础，但调节这些相互作用的分子机制却鲜为人知。我们在上一次对酿酒酵母的资助周期中的发现使我们处于有利的地位，可以解决与ORC在DNA复制启动中的角色相关的两个关键问题。首先，我们发现已经建立的酵母ORC-DNA界面不足以解释ORC与很大一部分起源的结合，并且ORC-染色质相互作用对萌芽酵母的起源选择的贡献比以前所认识的更大。因此，对于后生动物，ORC-染色质界面对于ORC在萌芽酵母中选择DNA复制起点是重要的。我们已经确定了“依赖于染色质”的酵母起源，这将使我们能够使用酵母强大的实验工具来定义ORC-染色质界面的分子细节，从而为ORC如何选择染色体内的DNA复制起源提供新的见解。染色质、其调节因子和真核生物中的DNA复制蛋白之间的高度保守性意味着我们在发芽酵母中定义的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/或有助于操纵该界面以改善人类健康。其次，来自各种系统的越来越多的证据表明，ORC与染色体和起源之间的关系比通常所描述或理解的更具活力。此外，数据表明，这些动力学与体内的起源调控有关。这些数据和我们最近对染色质依赖起源的功能的观察使我们得出了以下假设：由单个起源的ORC-染色质和ORC-DNA相互作用的差异引起的ORC起源动力学的差异，有助于调节用于复制真核基因组的100‘S到1000’S起源之间存在的起源激活的差异。我们将检验这一假说，从而对ORC-起源相互作用如何调节体内起源激活这一基本但相对未被探索的问题获得新的和重要的见解。

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.