Structural mechanism of DNA segregation by the pSK41 par system

描述（由申请人提供）：每一个物种的生存都依赖于基因信息的忠实遗传。这一过程的关键是染色体和质粒在细胞分裂时向子细胞的准确移动和定位。本建议的最终目标是在原子水平上阐明DNA分离的基本机制。质粒分割（par）系统是研究DNA分割分子机制的优秀模型系统，因为它们只需要三个组成部分：着丝粒位点和蛋白质，聚合物形成蛋白和着丝粒结合蛋白。人们最了解的par系统包含聚合物形成蛋白ParM和着丝粒结合蛋白ParR。本研究以金黄色葡萄球菌多药耐药质粒pSK41为研究对象，建立了一个这样的par系统。在分裂的第一步，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。然后，ParM- atp被招募到配对的凝体中，刺激ParM丝的形成。不断生长的ParM丝最终将成对的质粒分开。仍有待解决的重要问题是，ParR与其着丝粒结合形成何种类型的大型核蛋白复合体，这是如何招募ParM的，以及ParM是如何被ATP和凝体刺激形成细丝的？此外，pSK41系统在par系统中是独一无二的，除了ParR，它还需要一种新的染色体编码蛋白ArtA来完成par转录的自动调节。我们最近对pSK41 ParR与其着丝粒结合的结构的测定揭示了segrosome的第一个结构，并对segrosome如何招募和稳定丝的形成做出了重要的预测。在这项拨款提案中，我们将以最近的进展为基础，全面阐明pSK41分区及其调控，具体目标如下：(1)通过确定arda - dna复合体的结构，全面阐明pSK41 par操纵子的转录调控机制(2)通过对pSK41 ParM的关键构象状态（apoParM、ParM- adp和ParM- atp）以及parr -着丝粒-ParM- amp - pcp的结构进行结构和生化研究，阐明质粒分离的机制。我们还将阐明ParM丝的结构，并利用电子显微镜、生物化学和细胞研究来研究它与凝血体的相互作用。重要的是，藏在金黄色葡萄球菌中的pSK41对多种抗生素产生了耐药性，包括最后一种药物万古霉素。这种耐多药金黄色葡萄球菌正成为严重威胁人类健康的毒株。事实上，最近的报告表明，在美国，耐多药金黄色葡萄球菌导致的死亡人数已经超过了艾滋病毒/艾滋病。因此，本提案中描述的工作将通过靶向维持多重耐药决定因子所需的必需par蛋白和复合物，为针对此类多重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：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。pSK41质粒，藏于金黄色葡萄球菌中，携带许多这些多重耐药基因。质粒的保留要求它在细胞分裂过程中被准确分离；依赖于其par系统的进程。因此，通过该par系统了解DNA分离的结构基础，将为针对含有耐多药金黄色葡萄球菌菌株的pSK41提供几个潜在的治疗干预点。

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.