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: 10277063
• 负责人: HYEONGJUN KIM
• 金额: $ 36.34万
• 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/10277063
• 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; Genetic Translation; Genome Stability; Goals; Hydrolysis; Immunoprecipitation; In Vitro; Laboratories; Link; Maintenance; Malignant Neoplasms; Measurement; Modeling; Molecular; Nature; Process; Proteins; Replication Origin; Research; Role; Site; Slide; Structure; Surface Plasmon Resonance; System; Techniques; Theoretical model; Thinking; Variant; Work; base; biophysical techniques; cofactor; daughter cell; genetic information; genome integrity; in vivo; novel; programs; protein complex; protein function; recruit; single molecule; 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.

项目概要/摘要 细胞生物学的一个基本问题是理解 DNA 是如何通过密集的压缩来构造的。 包装细胞环境，并准确地传递给子细胞。 是动态准确组织染色体的关键因素，直接影响复制， 因此，包括各种癌症在内的许多疾病都是相关的。 在大多数细菌中，ParABS 分配系统和染色体相关蛋白发生故障。 染色体结构维持 (SMC) 蛋白复合物是染色体的主要贡献者 ParABS 系统由 ATP 酶变体 ParA、短回文 DNA 组成。 序列 parS 和 parS 结合蛋白 ParB parS 位点位于细菌起源附近。 染色体生物学领域的一个长期难题是 ParB 蛋白不仅被发现。 在 PAS 站点上，但也关联广泛的（10-20 kb）侧翼区域 - 这种现象称为扩散。 归因于 ParB 蛋白桥接 DNA 不同片段的能力，从而允许长距离 一种新的思维方式源自最近的发现，即 ParB 蛋白不仅仅是 DNA 结合。 蛋白质也是一种新型 CTPase 酶，有人提出胞苷三磷酸 (CTP) 与 ParS 结合。 及其随后的水解循环驱动 ParS 自加载到 parS 位点上并随后滑走 然而，这种“夹紧和滑动”模型本身在解释体内时存在局限性。 染色体免疫沉淀数据 ParB 蛋白的另一个重要作用是它们招募 SMC 蛋白。 然而，人们对 SMC 蛋白的募集知之甚少。 一旦被招募，细菌 SMC 就会通过主动挤压 DNA 环来组织 DNA。 需要证明可以解释染色体结构许多方面的简单机制 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 未来五年，PI实验室将解决ParB底层的突出问题。 利用他的单分子方法和新方法研究细菌 SMC 工作机制及其相互作用 获得了基于表面等离子共振（SPR）的专业知识，可以从这些蛋白质中提取信息。 在传统的生物学方法中，由于同时测量的性质，可能会被平均掉 我们的方法有望揭示隐藏的机制。 细节前所未有。体外结果将通过体内测定和理论模型得到证实。 拟议的工作将为未来其他 DNA-蛋白质相互作用研究铺平道路。 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 维持基因组的稳定性和动态。