Structural mechanism of DNA segregation by the pSK41 par system

描述（申请人提供）：每个物种的生存都依赖于遗传信息的忠实继承。这一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。这项提案的最终目标是在原子水平上阐明DNA分离的基本机制。质粒分配（PAR）系统是研究DNA分配的分子机制的极好模型系统，因为它们只需要三个组分：着丝粒位点和蛋白质，即聚合物形成蛋白和着丝粒结合蛋白。最好理解的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。该建议集中于来自金黄色葡萄球菌多药耐药质粒pSK 41的一个这样的par系统。在分裂的第一步中，ParR与着丝粒合作结合，形成一个大的包裹的核蛋白复合物，称为segrosome。ParM-ATP然后被募集到配对的segrosomes，刺激ParM丝形成。不断生长的ParM细丝最终将成对的质粒分开。仍然有待解决的重要问题是什么类型的大型核蛋白复合物是由ParR结合到其着丝粒，这是如何用于招募ParM和ParM是如何刺激ATP和segrosome形成细丝？此外，pSK 41系统是par系统中的独特之处在于，除了ParR，它需要一种新的染色体编码的蛋白质，阿尔塔，用于完全的par转录自动调节。我们最近对pSK 41 ParR与其着丝粒结合的结构测定揭示了segrosome的第一个结构，并对segrosome如何招募和稳定细丝形成做出了重要的预测。在本资助申请中，我们将基于最近的进展，全面阐明pSK 41分区及其调控，具体目标如下：（1）通过确定ArtA-DNA复合物的结构，充分阐明pSK 41 par操纵子的转录调节机制（2）通过对pSK 41 ParM关键构象状态的结构和生化研究，阐明质粒分离的机制（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，pSK 41在S.金黄色葡萄球菌对多种抗生素产生耐药性，包括最后的药物万古霉素。这种多重耐药的S.金黄色葡萄球菌菌株正成为人类健康的严重威胁。事实上，最近的报道表明，多药耐药的S。金黄色葡萄球菌现在在美国杀死的人比艾滋病毒/艾滋病更多。因此，在这项建议中描述的工作将提供潜在的点，治疗干预对这种多药耐药的S。通过靶向维持多药耐药决定簇所需的必需PAR蛋白和复合物来抑制金黄色葡萄球菌菌株。公共卫生相关性：耐多药金黄色葡萄球菌现在在美国杀死的人比艾滋病毒/艾滋病更多。pSK 41质粒存在于S.金黄色葡萄球菌携带许多这种多药耐药基因。这种质粒的保留要求它在细胞分裂过程中被精确分离;这一过程依赖于其par系统。因此，了解这种par系统的DNA分离的结构基础，将提供几个潜在的治疗干预点对pSK 41窝藏多药耐药S。金黄色葡萄球菌菌株。

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.