Mechanisms for recruitment and function of metazoan replication initiation factors

项目摘要 生物体的生存有赖于染色体DNA的及时准确复制。总而言之， 在生命结构域中，DNA复制(或启动)的开始依赖于结合特定启动子蛋白 基因组位置，称为复制起点，并帮助将复制解旋酶加载到DNA上。在真核生物中， 启动子是起源识别复合体(ORC)，它招募和沉积McM2-7解旋酶马达 模组到DNA上，对起源进行“许可”。尽管核心复制启动因子在大多数情况下是保守的 在真核生物和芽殖酵母中都得到了很好的研究，在起源识别方面存在着重要的差异 以及对酿酒酵母和高等真核生物之间的原产地许可的规定。数不胜数 因此，后生动物系统中关于核心和后生动物来源、征聘和职能的问题仍然存在。 辅助DNA复制起始因子，以及染色质相关蛋白对 这些事件。在这项提案中，我们的目标是在后生动物系统中的机械水平上定义ORC- 依赖的DNA重塑有助于Mcm2-7的负载，疾病相关突变如何改变启动子 活性，以及ORC伙伴蛋白如何通过以下方式促进染色体招募和启动子的功能 整合生化、结构和基于细胞的方法。这项工作的发现将具有广泛的意义 对多个科学领域的影响，因为它们不仅有助于为起源规范生成模型， 后生动物的原产地加工和原产地许可，但也有助于我们对DNA的理解--以及 依赖染色质的大分子机器。我们的研究也具有重要的生物医学意义，因为 未能精确复制染色体DNA导致基因组不稳定，这反过来又支撑了许多 人类疾病，包括癌症。因此，从长远来看，我们努力的结果有可能 为开发新的治疗策略提供重要的起点。

Project Summary The survival of living organisms depends on the timely and accurate duplication of chromosomal DNA. In all domains of life, the onset of DNA replication (or initiation) relies on dedicated initiator proteins that bind genomic sites, termed replication origins, and help load replicative helicases onto DNA. In eukaryotes, the initiator is the origin recognition complex (ORC), which recruits and deposits the Mcm2-7 helicase motor module onto DNA to ‘license’ origins. Although core replication initiation factors are conserved across most eukaryotes and are well-studied in budding yeast, important differences exist with respect to origin recognition and the regulation of origin licensing between S. cerevisiae and higher eukaryotes. Numerous outstanding questions therefore remain in the metazoan system regarding origin recruitment and function of core and accessory DNA replication initiation factors, and the specific contributions of chromatin-associated proteins to these events. In this proposal, we aim to define at a mechanistic level in metazoan systems how ORC- dependent DNA remodeling contributes to Mcm2-7 loading, how disease-linked mutations alter initiator activities, and how ORC-partner proteins promote chromosomal recruitment and function of the initiator, by integrating biochemical, structural, and cell-based approaches. The findings from this work will have broad implications for multiple scientific fields, as they will not only help generate models for origin specification, origin processing, and origin licensing in metazoans, but also contribute to our understanding of DNA- and chromatin-dependent macromolecular machines. Our studies are also of significant biomedical relevance since the failure to precisely replicate chromosomal DNA causes genome instability, which in turn underpins many human diseases, including cancer. In the long-term, the outcomes of our efforts thus have the potential to provide important starting points for the development of novel therapeutic strategies.