Structural investigation of human ORC: a key determinant of DNA origin selection

摘要 基因组复制是所有生命形式中必不可少的事件。DNA复制起始于特定位点（称为 复制起点）以促进基因组的适当复制。人类的 调节DNA复制起点位置的决定因素相对来说还没有得到解决。这里我们将 从结构上研究在人类中建立复制起点的关键决定因素， DNA起源选择机制建立复制起点的最初因素是起点 识别复合物（ORC）。ORC将蛋白质CDC 6招募到DNA复制起点以形成前- 复制复合体（pre-RC）。考虑到ORC基因的突变， 可能导致有害的影响，如Meier-Gorlin综合征和癌症， 复制的我们将通过结合研究，如电泳，研究ORC·CDC 6·DNA相互作用。 迁移率变动（EMSA）和Förster共振能量转移（FRET）测定。ORC·CDC 6·DNA复合物 将通过冷冻电子显微镜（cryo-EM）技术进行进一步分析。研究表明，ORC和 CDC 6被募集到沿着环状结构的染色质的既定位置。的染色质区域 主动转录由称为核小体的组蛋白复合物组成，其沿DNA间歇性地沿着定位， 这些核小体影响ORC的建立并因此影响复制起点的选择。组蛋白 亚基经历许多影响ORC与复合物结合的翻译后修饰。我们将 通过先进的冷冻EM方法在结构上研究前RC·核小体相互作用，以确定第一个 基因组复制的一步。将优化预RC和核小体重建以产生稳定的细胞。 和均匀的样品。将开发现代荧光标记方法来分析结合 配合物的性质。翻译后修饰，例如磷酸化和甲基化，将被 以确定对ORC和核小体识别的影响。结果从结构和 结合研究将支持ChIP-Seq检测的开发，以绘制ORC和复制起点 基因组定位深入了解ORC·CDC 6·核小体相互作用是解开 DNA起源选择的机制，并将为药物化合物的设计提供见解， 逆转不完全复制的影响。我的长期目标是成为一个 一个独立的研究实验室，对DNA复制的结构生物学进行高影响研究 和染色质调节。NIH F32奖学金将提供巨大的学习和研究支持， 这个目标此外，冷泉港实验室还举办国家会议（例如：真核DNA 复制和基因组维护），课程（例如：冷冻电子显微镜，3月）和研讨会 （领导力）对我的科学发展来说是例外的。

Abstract Genome replication is an essential event in all forms of life. DNA replication is initiated at specific sites (termed origins of replication) along chromosomes to facilitate appropriate duplication of the genome. In humans, the determinants that regulate the location of DNA replication origin are relatively unresolved. Here we will structurally investigate key determinants that establish origins of replication in humans to define the mechanism of DNA origin selection. The initial factor that establishes the origin of replication is the origin recognition complex (ORC). ORC recruits the protein CDC6 to the DNA origin of replication to form the pre- replicative complex (pre-RC). The complex is essential for replication, considering mutations in ORC genes can lead to deleterious effects, such as Meier-Gorlin Syndrome and cancer resulting from incomplete replication. We will investigate ORC•CDC6•DNA interactions through binding studies, such as electrophoretic mobility shift (EMSA) and Förster resonance energy transfer (FRET) assays. The ORC•CDC6•DNA complex will be further analyzed through cryoelectron microscopy (cryo-EM) techniques. Studies have shown ORC and CDC6 are recruited to established locations along variably structured chromatin. The chromatin regions of active transcription consist of histone complexes, called nucleosomes, positioned intermittently along DNA and these nucleosomes influence ORC establishment and therefore replication origin selection. The histone subunits undergo many posttranslational modifications that influence ORC binding to the complex. We will structurally investigate pre-RC•nucleosome interactions through advanced cryo-EM methods to define the first step in genome replication. The pre-RC and nucleosome reconstitution will be optimized to generate stable and homogenous samples. Modern fluorescent labelling-approaches will be developed to analyze the binding properties of the complexes. Posttranslational modifications, such as phosphorylation and methylation, will be addressed to determine the influence on ORC and nucleosome recognition. The results from the structural and binding studies will support development of a ChIP-Seq assay to map out the ORC and replication origin genome location. An in depth understanding of ORC•CDC6•nucleosome interactions are key to unraveling the mechanism of DNA origin selection and will provide insight for the design of pharmaceutical compounds that reverse the effects of incomplete replication. My long-term goal is to become the principal investigator of an independent research laboratory that conducts high impact studies on the structural biology of DNA replication and chromatin regulation. The NIH F32 fellowship will provide immense learning and research support towards this goal. In addition, the Cold Spring Harbor Laboratory harbors national meetings (ex: Eukaryotic DNA Replication & Genome Maintenance), courses (ex: Cryoelectron Microscopy, March), and workshops (Leadership) that are exceptional for my scientific development.