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 细胞生物学中的一个基本问题是理解DNA是如何通过致密的 包装细胞环境，并准确地传递到子细胞。染色体相关蛋白 是动态准确组织染色体的关键因素，直接影响复制， 基因信息的转录和翻译。因此，包括各种癌症在内的许多疾病都是相互关联的 与染色体相关蛋白的故障有关。在大多数细菌中，ParABS分配系统和 染色体的结构维持(SMC)蛋白复合体是染色体的主要贡献者 种族隔离和组织。ParABS系统由ATPase变异体ParA、短回文DNA组成 序列PARS和PARS结合蛋白PARB。PARS位置位于细菌起源的附近 复制。染色体生物学领域的一个长期难题是，PARB蛋白不仅存在于 在PARS位点上，但也关联广泛的(10-20kb)侧翼区域-一种称为扩散的现象。它 已经被归因于PARB蛋白连接不同DNA片段的能力，这使得长距离 互动。一种新的思维方式源于最近的发现，即PARB蛋白不仅仅是DNA结合 蛋白质，也是一种新型的CTPase酶。有人认为，胞苷三磷酸(CTP)与PARs结合 其随后的水解循环驱动PARs自身加载到PARs位置并随后滑动离开 从装货地点运来。然而，这种“夹住和滑动”模型本身在解释活体模型方面存在局限性。 染色体免疫沉淀数据。PARB蛋白的另一个关键作用是它们招募SMC蛋白 复制起始点附近的复合体。然而，对SMC蛋白的募集知之甚少。 机制。一旦被招募，细菌SMC被认为通过主动挤出DNA环来组织DNA。这 需要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复合体。PI拥有近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。在未来五年，PI的实验室将解决潜在的PARB的突出问题 和细菌SMC的作用机制及其相互作用 获得基于表面等离子体共振(SPR)的专业知识。人们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，可能会取平均值 多种蛋白质(整体测量)。我们的方法将有望发现隐藏的机制 史无前例的细节。体外实验结果将通过基于体内的分析和理论建模得到证实。 这项拟议的工作将为未来的其他DNA-蛋白质相互作用研究铺平道路。中国的长期目标是 Pi的研究计划是阐明不同的DNA结合蛋白及其辅助因子是如何协同作用的 保持基因组的稳定性和动态平衡。