Molecular basis of bacterial chromosome segregation and organization

细菌染色体分离和组织的分子基础

• 批准号: 10799361
• 负责人: HYEONGJUN KIM
• 金额: $ 14.09万
• 依托单位: UNIVERSITY OF TEXAS RIO GRANDE VALLEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799361
• 关键词: ATP phosphohydrolase; Accounting; Bacteria; Bacterial Chromosomes; Binding; Binding Proteins; Biological Assay; Biology; CTPase; Cellular biology; Chromatin Loop; Chromosome Segregation; Chromosome Structures; Chromosomes; Closure by clamp; Complex; Cytidine; DNA; DNA Sequence; DNA Structure; DNA-Binding Proteins; DNA-Protein Interaction; Data; Disease; Environment; Enzymes; Future; Genetic Transcription; Genome Stability; Goals; Hydrolysis; Immunoprecipitation; In Vitro; Laboratories; Link; Maintenance; Malignant Neoplasms; Measurement; Modeling; Molecular; Nature; Proteins; Replication Origin; Research; Role; Site; Slide; Surface Plasmon Resonance; System; Techniques; Theoretical model; Thinking; Variant; Work; cofactor; daughter cell; genetic information; in vivo; novel; programs; protein complex; recruit; single molecule; translational genetics; tripolyphosphate

PROJECT SUMMARY/ABSTRACT A fundamental problem in cell biology is understanding how DNAs are structured by compaction in the densely packed cellular environment, and accurately passed down to daughter cells. Chromosome-associated proteins are key factors in dynamically and accurately organizing chromosomes, and directly influence the replication, transcription, and translation of genetic information. As such, many diseases including various cancers are linked to malfunctioning of chromosome-associated proteins. In a majority of bacteria, ParABS partitioning system and structural maintenance of chromosomes (SMC) protein complex are main contributors for chromosome segregation and organization. The ParABS system is composed of ATPase variant ParA, short palindromic DNA sequence parS, and parS-binding protein ParB. The parS sites are located in the vicinity of bacterial origin of replication. A longstanding conundrum in the chromosome biology field is that ParB proteins are not only found on the parS sites but also associate extensive (10-20 kb) flanking regions – a phenomenon termed spreading. It had been attributed to the ability of ParB protein to bridge different segments of DNA, that allows long-distance interactions. A new way of thinking derived from recent discoveries that ParB protein is not merely a DNA binding protein but also a novel CTPase enzyme. It was proposed that cytidine triphosphate (CTP) binding to the ParS and its subsequent hydrolysis cycle drives self-loading of ParS onto parS sites and subsequent sliding away from the loading sites. However, this “clamp and sliding” model alone has limitations in accounting for in vivo chromosome immunoprecipitation data. Another critical role of ParB proteins is that they recruit SMC protein complex to the vicinity of the replication origin. However, little has been known about the SMC protein recruitment mechanism. Once recruited, bacterial SMC is thought to organize DNAs by actively extruding DNA loops. This simple mechanism that can explain many aspects of chromosome structuring is required to be demonstrated with bacterial SMC complex. The PI has almost 15 years of single-molecule techniques expertise and his lab is devoted to elucidating the mechanisms of various DNA-binding proteins and their impacts on chromosome structure. During the next five years, the PI’s laboratory will tackle the outstanding problems of underlying ParB and bacterial SMC working mechanisms and their interplays utilizing his single-molecule approaches and newly acquired surface plasmon resonance (SPR)-based expertise. Information one could extract from those proteins in traditional biology approaches is possibly averaged out due to the nature of simultaneous measurements of multiple proteins (ensemble measurements). Our approach will be expected to uncover hidden mechanisms with unprecedented details. The in vitro results will be corroborated by in vivo-based assays and theoretical modeling. The proposed work will pave the way for other future DNA-protein interaction studies. The long-term goal of the PI’s research program is to elucidate how different DNA-binding proteins and their cofactors cooperate to maintain the genome stability and dynamics.

项目摘要/摘要 细胞生物学的一个基本问题是了解DNA如何通过密集的压实构造 挤满了细胞环境，并准确地传递给子细胞。染色体相关蛋白 是动态和准确组织染色体的关键因素，并直接影响复制， 转录和遗传信息的翻译。因此，许多疾病包括各种癌症 与染色体相关蛋白的功能故障。在大多数细菌中，parabs分区系统和 染色体的结构维持（SMC）蛋白质复合物是染色体的主要贡献者 隔离和组织。 Parabs系统由ATPase变体Para组成 序列PAR和PARS结合蛋白PAR。 pars位置位于细菌起源附近 复制。染色体生物学领域中长期存在的难题是PARB蛋白不仅被发现 在pars站点上，但也将广泛的（10-20 kb）侧翼区域关联 - 一种现象术语扩散。它 已归因于PARB蛋白桥接不同DNA段的能力，这可以长距离 互动。最近发现的一种新的思维方式，即PARB蛋白不仅是DNA结合 蛋白质也是一种新型的CTPase酶。有人提出，三磷酸胞苷（CTP）与PARS结合 随后的水解周期将PARS自动加载到PARS位点，然后滑行 从加载站点。但是，单独的“夹具和滑动”模型在体内会计局限 染色体免疫沉淀数据。 PARB蛋白的另一个关键作用是它们募集SMC蛋白 复杂起源附近的复杂性。但是，关于SMC蛋白募集的知之甚少 机制。一旦招募，细菌SMC被认为通过主动挤出DNA环会组织DNA。这 可以证明可以解释染色体结构的许多方面的简单机制 与细菌SMC复合物。 PI具有将近15年的单分子技术专业知识，他的实验室是 致力于阐明各种DNA结合蛋白的机制及其对染色体的影响 结构。在接下来的五年中，PI的实验室将解决基本的问题 和细菌SMC的工作机制及其相互作用，利用他的单分子方法和新的 获得的表面等离子体共振（SPR）的专业知识。可以从这些蛋白质中提取的信息 在传统的生物学方法中，由于同时测量的性质，很可能会平均 多种蛋白质（集合测量）。我们的方法将有望与 空前的细节。基于体内的测定和理论建模将证实体外结果。 拟议的工作将为其他未来的DNA蛋白相互作用研究铺平道路。长期目标 PI的研究计划是阐明不同的DNA结合蛋白及其辅助因子如何合作 保持基因组稳定性和动力学。