Structural mechanism of DNA segregation by the pSK41 par system

描述(申请人提供)：每个物种的生存都有赖于遗传信息的忠实传承。这一过程的关键是染色体和质粒在细胞分裂时准确地移动和定位到子代细胞。这一提议的最终目标是在原子水平上阐明DNA分离的基本机制。质粒分配(PAR)系统是研究DNA分配分子机制的优秀模型系统，因为它们只需要三个组分：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。这项建议集中在金黄色葡萄球菌多药耐药质粒pSK41的一个这样的PAR系统。在分割的第一步，PARR与着丝粒协同结合，形成一个大的、包裹着丝粒的核蛋白复合体，称为segroome。然后，PARM-ATP被招募到成对的底物中，刺激PARM细丝的形成。不断生长的帕姆细丝最终将成对的质粒分开。有待解决的关键问题是，PARR与着丝粒结合形成了什么类型的大核蛋白复合体，这如何起到招募PARM的作用，以及PARM是如何被ATP和丝状体刺激形成细丝的？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、染色体编码的蛋白质ARTA来实现完整的PAR转录自动调节。我们最近对与着丝粒结合的pSK41ParR的结构进行了测定，揭示了丝状体的第一种结构，并对丝状体如何招募和稳定丝状体做出了重要的预测。我们还将阐明Parm细丝的结构，并使用电子显微镜、生物化学和细胞研究来研究它与segroome的相互作用。重要的是，含有金黄色葡萄球菌的pSK41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，耐多药金黄色葡萄球菌导致的死亡人数超过了艾滋病毒/艾滋病。因此，这项建议中描述的工作将通过靶向维持多药耐药决定因素所需的基本PAR蛋白和复合体，为此类多药耐药金黄色葡萄球菌菌株提供潜在的治疗干预点。与公共卫生相关：在美国，多重耐药金黄色葡萄球菌导致的死亡人数超过了艾滋病毒/艾滋病。含有金黄色葡萄球菌的pSK41质粒携带许多这种多重耐药基因。这种质粒的保留要求它在细胞分裂过程中被准确分离；这一过程取决于它的PAR系统。因此，了解这种PAR系统分离DNA的结构基础将为治疗携带多重耐药金黄色葡萄球菌菌株的pSK41提供几点潜在的治疗干预。

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.