Mechanisms for recruitment and function of metazoan replication initiation factors

项目摘要 生物体的生存依赖于染色体DNA的及时和准确的复制。在所有 DNA复制的开始（或起始）依赖于专门的起始蛋白， 基因组位点，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， 起始物是起始识别复合物（origin recognition complex，ORC），其募集并沉积Mcm 2 -7解旋酶马达 模块到DNA“许可证”的起源。虽然核心复制起始因子在大多数 在真核生物和芽殖酵母中得到充分研究，在起源识别方面存在重要差异 以及中美之间的原产地许可规则。酿酒酵母和高等真核生物。无数优秀 因此，问题仍然存在于后生动物系统的起源，招聘和功能的核心， 辅助DNA复制起始因子，以及染色质相关蛋白对 这些事件。在这个建议中，我们的目标是在后生动物系统中的机制水平上定义ORC- 依赖性DNA重塑有助于Mcm 2 -7加载，疾病相关突变如何改变启动子 活性，以及ORC-伴侣蛋白如何促进染色体募集和启动子的功能， 整合了生物化学、结构和细胞方法。这项工作的结果将具有广泛的 对多个科学领域的影响，因为它们不仅有助于生成原产地规范模型， 起源加工和起源许可的后生动物，但也有助于我们了解DNA-和 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 不能精确复制染色体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，我们努力的成果有可能 为开发新的治疗策略提供了重要的起点。

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.