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: 7728001
• 负责人: Maria Schumacher
• 金额: $ 34.65万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7728001
• 关键词: AIDS/HIV problem; Address; Adenine Nucleotides; Adopted; Affect; Antibiotics; Applications Grants; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biological Models; Cell division; Centromere; Charge; Chimeric Proteins; Chromosome Positioning; Codon Nucleotides; Complex; Crystallization; DNA; Data; Electron Microscopy; Electrostatics; Elements; Escherichia coli; Filament; Fluorescence Polarization; Foundations; Genes; Genetic; Genetic Materials; Genetic Transcription; Goals; Health; Homeostasis; Human; Image; In Vitro; Individual; Link; Maintenance; Mediating; Modeling; Molecular; Molecular Conformation; Motor; Movement; Multi-Drug Resistance; Multidrug Resistance Gene; Mutation; Nucleoproteins; Operon; Pharmaceutical Preparations; Plasmids; Play; Polymers; Process; Property; Proteins; Recruitment Activity; Regulation; Reporting; Resistance; Resolution; Resort; Role; Site; Staphylococcus aureus; Structure; Surface; Synthetic Genes; System; Tandem Repeat Sequences; Testing; Therapeutic Intervention; Transcriptional Regulation; Vancomycin; Work; base; daughter cell; design; image reconstruction; in vivo; insight; killings; mutant; novel; nucleic acid structure; promoter; protein complex; public health relevance; segregation

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。这一过程至关重要的是染色体和质粒对细胞分裂的子细胞的准确运动和定位。该提案的最终目标是在DNA隔离的原子水平上阐明基本机制。质粒分区（PAR）系统代表了研究DNA分区的分子机制的出色模型系统，因为它们仅需要三个组件：一个丝粒位点和蛋白质，一种组成的聚合物蛋白和共粒结合蛋白。最好理解的PAR系统包含称为PARM的聚合物形成蛋白，称为PARR。该提案的重点是金黄色葡萄球菌多药抗性质粒PSK41的一个这样的PAR系统。在分区的第一步中，Parr与Centromere合作结合，形成一个称为脱绿色的大型包裹的核蛋白复合物。然后将PARM-ATP募集到配对的透明体中，从而刺激Parm细丝形成。越来越多的Parm细丝最终将配对的质粒拆开。尚待解决的重要问题是，parr结合其与中心粒形成了哪种类型的大核蛋白复合物，这是如何用于募集PARM的方式以及如何刺激PARM以ATP和SEPROSOM的形成细丝？此外，PSK41系统在PAR系统之间是独特的，因为除了PARR之外，它还需要一种新型的，染色体编码的蛋白ARTA，以进行全PAR转录自动调节。我们最近与其中心粒结合的PSK41 PARR的结构确定揭示了脱落的第一个结构，并就表会体如何募集和稳定细丝形成做出了重要的预测。在这项赠款的提案中，我们将基于这一最新进展，以全面阐明PSK41分区及其对以下具体目的进行调节：（1）完全阐明PSK41 PAR操纵子的转录机制，通过确定ARTA-DNA复合物的结构（2）通过质量分离的结构和生物学研究的机制来确定ARTA-DNA复合物的结构（2） PARM-ADP和PARM-ATP）以及PARR-CENTROMERE-PARM-PARM-AMP-PCP的结构。我们还将使用电子显微镜，生物化学和细胞研究来阐明PARM细丝的结构，并检查其与腹腔体的相互作用。重要的是，在金黄色葡萄球菌中设有的PSK41赋予了对多种抗生素的耐药性，包括上一位度假胜地Vanomycin的药物。这种多药抗性金黄色葡萄球菌菌株正对人类健康构成严重威胁。实际上，最近的报告表明，抗多药的金黄色葡萄球菌现在杀死了美国更多的人，而不是艾滋病毒/艾滋病。因此，本提案中描述的工作将通过靶向维持多药耐药性确定因素所需的必需PAR蛋白和复合物来为这种多药抗性金黄色葡萄球菌菌株提供治疗干预的潜在点。公共卫生相关性：多金黄色葡萄球菌的多药抗药性现在杀死了美国的人数多于艾滋病毒/艾滋病。在金黄色葡萄球菌中含有的PSK41质粒带有许多这些多药抗性基因。保留该质粒要求在细胞分裂过程中准确隔离它。一个取决于其PAR系统的过程。因此，通过该PAR系统了解DNA分离的结构基础将提供一些潜在的治疗干预措施，以针对具有抗多重金黄色葡萄球菌菌株的PSK41。