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复制在特定的站点(称为 复制的起源)，以促进基因组的适当复制。在人类中， 调控DNA复制起始点位置的决定因素相对来说还没有解决。在这里，我们将 从结构上研究在人类中建立复制起源的关键决定因素，以定义 DNA碱基选择的机制。建立复制来源的初始因素是来源 识别复合体(ORC)。ORC将CDC6蛋白招募到复制的DNA起始位置，以形成前 复制复合体(前RC)。考虑到ORC基因的突变，这种复合体对于复制是必不可少的 会导致有害的影响，如梅尔-戈林综合征和不完全导致的癌症 复制。我们将通过结合研究来研究ORC·CDC6·DNA的相互作用，例如电泳法 迁移率位移(EMSA)和Förster共振能量转移(FRET)分析。ORC·CDC6·DNA复合体 将通过低温电子显微镜(Cryo-EM)技术进行进一步分析。研究表明，兽人和 CDC6被招募到沿着可变结构的染色质的既定位置。的染色质区域 活性转录由组蛋白复合体组成，称为核小体，间歇性地位于DNA和 这些核小体影响ORC的建立，从而影响复制起点的选择。组蛋白 亚基经历了许多翻译后修饰，影响了ORC与复合体的结合。我们会 通过先进的冷冻-EM方法从结构上研究前RC·核小体相互作用，以定义第一 参与基因组复制。将优化Pre-RC和核小体重组，以产生稳定的 和同质样本。将开发现代荧光标记方法来分析结合 络合物的性质。翻译后修饰，如磷酸化和甲基化，将是 旨在确定对ORC和核小体识别的影响。来自结构和结构的结果 结合研究将支持芯片序列分析的发展，以绘制ORC和复制起点 基因组定位。深入了解ORC·CDC6·核小体相互作用是解开ORC·CDC6·核小体相互作用的关键 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名 对DNA复制的结构生物学进行高影响研究的独立研究实验室 和染色质调节。NIH F32奖学金将为以下方面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了国家会议(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非同寻常的。

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.