Structural mechanism of DNA segregation by the pSK41 par system

pSK41 par系统DNA分离的结构机制

• 批准号: 8236042
• 负责人: Maria Schumacher
• 金额: $ 32.9万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236042
• 关键词: Structural; mechanism; DNA; segregation; pSK41

DESCRIPTION (provided by applicant): The survival of every species depends on the faithful inheritance of genetic information. Essential to this process are the accurate movement and positioning of chromosomes and plasmids to daughter cells at cell division. The ultimate goal of this proposal is to elucidate at the atomic level, basic mechanisms of DNA segregation. Plasmid partition (par) systems represent excellent model systems to study the molecular mechanisms of DNA partition because they require only three components: a centromere site, and proteins, a polymer-forming protein and centromere-binding protein. The best understood par systems contain polymer-forming proteins called ParM and centromere-binding proteins called ParR. This proposal focuses on one such par system from Staphylococcus aureus multi-drug resistance plasmid pSK41. In the first step of partition, ParR binds cooperatively to the centromere to form a large, wrapped nucleoprotein complex called the segrosome. ParM-ATP is then recruited to paired segrosomes, stimulating ParM filament formation. The growing ParM filament ultimately pushes the paired plasmids apart. Vital questions that remain to be resolved are what type of large nucleoprotein complex is formed by ParR binding to its centromere, how this serves to recruit ParM and how ParM is stimulated to form filaments by ATP and the segrosome? In addition, the pSK41 system is unique among par systems in that, in addition to ParR, it requires a novel, chromosomally encoded protein, ArtA, for full par transcription autoregulation. Our recent structure determination of pSK41 ParR bound to its centromere reveal the first structure of a segrosome and make important predictions about how segrosomes recruit and stabilize filament formation. In this grant proposal we will build on this recent progress towards a full elucidation of pSK41 partition and its regulation with the following Specific Aims: (1) Fully elucidate the mechanism of transcription regulation of the pSK41 par operon by determining the structure of the ArtA-DNA complex (2) Clarify the mechanism of plasmid separation via structural and biochemical studies on the key conformational states of pSK41 ParM (apoParM, ParM-ADP and the ParM-ATP) as well as a structure of ParR-centromere-ParM-AMP-PCP. We will also elucidate the structure of the ParM filament and examine its interaction with the segrosome using electron microscopy, biochemistry and cellular studies. Importantly, pSK41, harbored in S. aureus, confers resistance to multiple antibiotics, including the drug of last resort, vancomycin. Such multi-drug resistant S. aureus strains are becoming a serious threat to human health. Indeed, recent reports indicate that multidrug resistant S. aureus now kills more individuals in the U.S. than HIV/AIDS. Thus, the work described in this proposal will provide potential points of therapeutic intervention against such multi-drug resistant S. aureus strains by targeting the essential par proteins and complexes required for maintenance of multi-drug resistance determinants. PUBLIC HEALTH RELEVANCE: Multidrug resistant Staphylococcus aureus now kills more individuals in the U.S. than HIV/AIDS. The pSK41 plasmid, which is harbored in S. aureus, carries many of these multi-drug resistant genes. Retention of this plasmid demands it be accurately segregated during cell division; a process dependent on its par system. Thus, understanding the structural basis for DNA segregation by this par system will provide several points of potential therapeutic intervention against pSK41 harboring multidrug resistant S. aureus strains.

描述（由申请人提供）：每个物种的生存都取决于遗传信息的忠实继承。此过程的关键是在细胞分裂时染色体和质粒向子细胞的准确移动和定位。该提案的最终目标是在原子水平上阐明 DNA 分离的基本机制。质粒分区 (par) 系统是研究 DNA 分区分子机制的优秀模型系统，因为它们只需要三个组件：着丝粒位点、蛋白质、聚合物形成蛋白和着丝粒结合蛋白。最了解的 par 系统包含称为 ParM 的聚合物形成蛋白和称为 ParR 的着丝粒结合蛋白。该提案重点关注金黄色葡萄球菌多重耐药质粒 pSK41 中的一个这样的 par 系统。在分区的第一步中，ParR 与着丝粒协同结合，形成一个大的、包裹的核蛋白复合物，称为segrosome。然后 ParM-ATP 被募集到配对的节粒体中，刺激 ParM 丝的形成。不断生长的 ParM 丝最终将配对的质粒推开。仍有待解决的重要问题是 ParR 与其着丝粒结合形成什么类型​​的大核蛋白复合物，它如何招募 ParM 以及 ParM 如何被 ATP 和 segrosome 刺激形成丝？此外，pSK41 系统在 par 系统中是独一无二的，因为除了 ParR 之外，它还需要一种新的染色体编码蛋白 ArtA 来实现完整的 par 转录自动调节。我们最近对与其着丝粒结合的pSK41 ParR的结构测定揭示了segrosome的第一个结构，并对segrosome如何募集和稳定丝形成做出了重要预测。在这项拨款提案中，我们将在最近取得的进展的基础上，全面阐明 pSK41 分区及其调控，具体目标如下：(1) 通过确定 ArtA- 的结构，充分阐明 pSK41 par 操纵子的转录调控机制。 DNA 复合物 (2) 通过对 pSK41 ParM（apoParM、ParM-ADP 和ParM-ATP) 以及 ParR-着丝粒-ParM-AMP-PCP 的结构。我们还将阐明 ParM 丝的结构，并使用电子显微镜、生物化学和细胞研究检查其与 segrosome 的相互作用。重要的是，金黄色葡萄球菌中携带的 pSK41 赋予对多种抗生素的耐药性，包括最后的药物万古霉素。这种多重耐药金黄色葡萄球菌菌株正在成为对人类健康的严重威胁。事实上，最近的报告表明，在美国，多重耐药金黄色葡萄球菌造成的死亡人数比艾滋病毒/艾滋病造成的死亡人数还要多。因此，本提案中描述的工作将通过靶向维持多药耐药决定簇所需的必需par蛋白和复合物，为针对此类多药耐药金黄色葡萄球菌菌株的治疗干预提供潜在的点。公共卫生相关性：目前，在美国，多重耐药金黄色葡萄球菌导致的死亡人数比艾滋病毒/艾滋病还要多。金黄色葡萄球菌中携带的 pSK41 质粒携带许多此类多重耐药基因。保留该质粒需要在细胞分裂过程中准确分离；依赖于其 par 系统的进程。因此，了解该 par 系统 DNA 分离的结构基础将为针对含有多重耐药金黄色葡萄球菌菌株的 pSK41 提供潜在的治疗干预。