Assembly and partition mechanism of Walker-box based segregation machinery

 描述（由申请人提供）：DNA分离或分区是生物学中最基本的过程之一，并确保遗传信息准确地从一代保留到下一代。虽然参与分配的分子已经在原核生物和真核生物中大量鉴定，但驱动这一过程的机制在原子水平上还不清楚。此外，目前对古细菌中的DNA分离知之甚少。该提案的总体目标是使用模型细菌和古细菌分区（PAR）系统来定义DNA分离中涉及的分子机制和原则。细菌质粒par系统代表了在原子水平上研究分离的优秀模型系统，因为它们最低限度地只需要3个元素，着丝粒，着丝粒结合蛋白（CBP）和NTR。最常见的PAR系统是所谓的I型，或基于Walker-box的系统。这种类型的PAR系统参与原核染色体和质粒的分区，并包括CBPs称为ParB和步行者型NTPases称为ParA。我们最近的结构和体内超分辨率研究的结合已经开始提供详细的原子以及细菌分离的动态了解。真核生物似乎使用与原核生物不同的机制，其中它们的着丝粒由“着丝粒核小体”标记，其含有CenpA代替组蛋白H3沿着H4、H2 A和H2 B。虽然这些系统看起来截然不同，但我们最近的研究揭示了生命3个领域中DNA分离之间的结构和潜在的功能联系。具体来说，我们的第一个archaa par系统，pNOB 8，初步表征表明，它采用了步行者盒NTR类似于原核生物所使用的，而我们的pNOB 8 ParB的初步结构表明，它包含一个类似于真核组蛋白CenpA蛋白的折叠。基于这些发现，我们假设，共享的结构和功能的原则可能存在于DNA分离机制在整个领域的生活。本提案的目标是提供详细的结构和功能解剖的DNA分离使用两个原型模型系统;细菌TP 228 PAR系统和古细菌pNOB 8 PAR系统。

 DESCRIPTION (provided by applicant): DNA segregation or partition is one of the most fundamental processes in biology and ensures that genetic information is accurately retained from one generation to the next. While the molecules involved in partition have largely been identified in prokaryotes and eukaryotes, the mechanisms that drive this process are not understood at the atomic level. Moreover, currently, very little is known about DNA segregation in archaea. The overarching goals of this proposal are to define the molecular mechanisms and principles involved in DNA segregation using model bacterial and archaeal partition (par) systems. Bacterial plasmid par systems represent excellent model systems to study segregation at an atomic level because they minimally require only 3 elements, a centromere, a centromere-binding protein (CBP) and an NTPase. The most common par system is the so-called type I, or Walker-box based system. This type of par system is involved in the partitioning of both prokaryotic chromosomes and plasmids, and includes CBPs called ParB and Walker-type NTPases called ParA. Our recent combination of structural and in vivo super-resolution studies has started to provide a detailed atomic as well as dynamic understanding of bacterial segregation. Eukaryotes appear to use machinery distinct from prokaryotes whereby their centromeres are marked by "centromeric nucleosomes", which contain CenpA in the place of histone H3 along with H4, H2A and H2B. Although these systems seem vastly disparate, our recent studies have revealed structural and, potentially, functional links between DNA segregation in the 3 domains of life. Specifically, our initial characterization of the first archaa par system, pNOB8, reveals that it employs a Walker-box NTPase similar to those used by prokaryotes while our preliminary structure of the pNOB8 ParB reveals that it contains a fold similar to the eukaryotic histone CenpA protein. Based on these findings, we hypothesize that shared structural and functional principles may exist in DNA segregation mechanisms across the domains of life. The goals of this proposal are to provide detailed structural and functional dissections of DNA segregation using two prototypical model systems; the bacterial TP228 par system and the archaeal pNOB8 par system.