Mechanisms of Chromosome Maintenance in Bacteria

细菌染色体维持机制

• 批准号: 10262055
• 负责人: DHRUBA K CHATTORAJ
• 金额: $ 61.41万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262055
• 关键词: AT Rich Sequence; Affinity; Aneuploidy; Animal Model; Antibiotics; Automobile Driving; Bacteria; Bacterial Infections; Bacterial Model; Binding; Biological Models; Biological Process; Cell Cycle; Cell division; Cells; Cholera; Chromosome Segregation; Chromosomes; Communication; Complex; DNA; DNA Binding; DNA Structure; Defect; Development; Diffuse; Dimerization; Disease; Dissociation; Dowries; Drug Design; Drug Targeting; Elements; Ensure; Escherichia coli; Eukaryota; Family; Fertilization; Generations; Genetic; Genetic Transcription; Genome; Growth and Development function; History of Medicine; Housekeeping Gene; In Vitro; Inherited; Life; Location; Maintenance; Malignant Neoplasms; Modeling; Molecular Chaperones; Mutate; Mutation; Opportunistic Infections; Oral; Organism; Pharmaceutical Preparations; Plasmids; Play; Process; Proliferating; Protein Analysis; Protein Region; Reaction; Rehydrations; Replication Initiation; Replication Origin; Reporting; Resistance development; Rewards; Role; Single-Stranded DNA; Sister; Site; Stress; Structure; Time; Torsion; Transact; Ursidae Family; Vibrio; Vibrio cholerae; antimicrobial; antimicrobial drug; cancer cell; chromosome replication; combat; daughter cell; dimer; human disease; improved; insight; melting; monomer; mutant; novel; prevent; protein function; protein protein interaction; segregation

Of the two V. cholerae chromosomes, the larger one (Chr1) carries most of the housekeeping genes and is considered the primary chromosome. The smaller chromosome (Chr2) seems to have evolved from a plasmid. Plasmids, although prevalent as extrachromosomal elements in bacteria, are rarely found integrated into the chromosome and driving the chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time in the cell cycle, whereas timely firing is the norm for chromosomal origins in all domains of life. Comparison of plasmid and Chr2 replication initiation mechanisms could thus be valuable to understand how the timing of a biological process has evolved from being random to be specific in the cell cycle. The timing of Chr2 replication depends on prior replication of a site, crtS, in Chr1. Our discovery of this site (in 2014), where the Chr2 initiator RctB also binds, demonstrated that chromosomes do actively communicate and encouraged studies to understand the mechanism of replication coordination between the two chromosomes in other labs. In eukaryotes, uncoordinated replication from different origins causes developmental abnormalities and cancer. Our progress in understanding replication of Chr2 and its coordination with that of Chr1 is reported below. 1. Communication between Chr1 and Chr2 for replication initiation: Identification of a novel check point control in bacteria. The location of crtS on Chr1 is such that it would replicate just before the time of Chr2 replication initiation. This affords a straight forward mechanism for communication: Chr1 replication initiates first. When the fork passes through crtS, it activates the bound RctB initiator molecules that triggers Chr2 replication. Replication of crtS thus relieves the check point that prevents Chr2 replication. The mechanistic details of crtS activation of Chr2 replication however remain enigmatic. The activity appears to be remodeling of RctB that improves specifically the initiator's origin binding activity. As expected, initiator binding to the origin is such a fundamental requirement that when RctB was mutated at random but selecting for its initiator function, the mutants remained responsive to crtS without exception. In other words, crtS is controlling some basic function of RctB that cannot be inactivated. It appears that origin binding is such an activity of the initiator. Last year, we showed that crtS activity depends on a global transcription regulator Lrp that also binds to crtS together with RctB. Our present studies indicate that there are at least two mechanisms by which Lrp could activate RctB binding: Direct protein-protein interaction by which Lrp bound to crtS can help load RctB there, and second by modifying crtS DNA structure which apparently increases RctB's affinity for crtS DNA. 2. Control of initiator function by dimerization. Protein function is often controlled by oligomerization. RctB dimerizes efficiently but several studies indicate that monomers are the active initiators. To get further insight on the role of dimerization, we made several structure-guided mutations in the dimerization domain of RctB. The mutants were variously defective in dimerization and the dimerization defect was inversely correlated with the initiator function. This indicated that dimerization is an inhibitory mechanism for replication initiation. These mutants have been rewarding for two reasons: They were no longer responsive to crtS, indicating that crtS could be activating RctB by dimer dissociation, and they were responsive to molecular chaperones, indicating that monomerization is not sufficient to relieve chaperone requirement. These results supports an alternate model that monomers need to be remodeled for them to be active in initiation. 3. Towards generation of Vibrio-specific antimicrobial agents: RctB, is conserved only in the Vibrio family and appears ideally suited for developing potential drugs specifically against Vibrios. In the case of cholera, although oral rehydration treatment is the mainstay, antimicrobial therapy becomes mandatory at times, and V. cholerae is no exception in developing resistance to multiple antibiotics. By systematic structure-function analysis of the protein we are trying to zeroing in on regions of the protein that are essential for replication initiation and, hence, survival of Vibrio. These regions can be specifically targeted for drug design. 4. Replication initiation of Chr2: Opening of the strands of replication origin. All transactions on DNA require strand-opening which is an energetically unfavorable reaction. How cells overcome this energy barrier for replication initiation is not clearly understood. The hypothesis is that initiator binding to the origin creates torsional stress on DNA that is released in a neighboring AT-rich region which is easier to melt. However, the melting needs to be stabilized by capturing at least one of the single strands, otherwise the stress would diffuse out of the origin. Evidence in favor of stabilization has been obtained in the Chr2 origin. We find that the Chr2 initiator RctB has single-stranded DNA binding activity, which is greatly stabilized in vitro by specific double stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single and double stranded sites. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin. Initiator binding to origin double-stranded sites thus plays a dual role by contributing to both in initiation of opening by DNA stressing and stabilization of the opening by capturing a single strand.

在两条霍乱弧菌染色体中，较大的一条（Chr1）携带大部分管家基因，被认为是主染色体。较小的染色体（Chr2）似乎是从质粒进化而来的。质粒酶虽然作为染色体外元件在细菌中普遍存在，但很少被发现整合到染色体中并驱动染色体复制。一个原因可能是质粒起源的触发通常不限于细胞周期中的特定时间，而及时触发是生命所有领域中染色体起源的标准。因此，质粒和Chr2复制起始机制的比较对于理解生物过程的时间是如何从随机演变为细胞周期中的特异性是有价值的。Chr2复制的时间取决于Chr1中的一个位点crtS的先前复制。我们在2014年发现了这个位点，其中Chr2启动子RctB也结合，这表明染色体确实积极交流，并鼓励研究了解其他实验室中两条染色体之间的复制协调机制。在真核生物中，来自不同起源的不协调复制导致发育异常和癌症。我们在理解Chr2的复制及其与Chr1的协调方面的进展报告如下。1. Chr1和Chr2之间用于复制起始的通信：细菌中新型检查点控制的鉴定。crtS在Chr1上的位置使得它将在Chr2复制开始之前复制。这提供了一种直接的通信机制：首先启动Chr1复制。当分叉穿过crtS时，它激活触发Chr2复制的结合的RctB起始分子。因此，crtS的复制解除了阻止Chr2复制的检查点。然而，crtS激活Chr2复制的机制细节仍然是谜。该活性似乎是RctB的重塑，其特异性地改善了起始物的来源结合活性。正如预期的那样，起始物与起始物的结合是这样一个基本要求，即当RctB随机突变但选择其起始物功能时，突变体无一例外地保持对crtS的响应。换句话说，crtS控制着RctB的一些基本功能，这些功能不能被灭活。看来，起源绑定是这样一个活动的发起人。去年，我们发现crtS活性依赖于一个全局转录调节因子Lrp，该因子也与RctB一起与crtS结合。我们目前的研究表明，至少有两种机制，Lrp可以激活RctB的结合：直接蛋白质-蛋白质相互作用，Lrp结合crtS可以帮助负载RctB，第二，通过修饰crtS DNA结构，这显然增加了RctB的亲和力crtS DNA。2.通过二聚作用控制引发剂的功能。蛋白质的功能通常由寡聚化控制。RctB有效地二聚化，但一些研究表明，单体是活性引发剂。为了进一步了解二聚化的作用，我们在RctB的二聚化结构域中进行了几次结构引导突变。突变体在二聚化方面有各种缺陷，并且二聚化缺陷与引发剂功能呈负相关。这表明二聚化是复制起始的抑制机制。这些突变体有两个原因：它们不再对crtS有反应，表明crtS可以通过二聚体解离激活RctB，它们对分子伴侣有反应，表明单体化不足以缓解伴侣的需求。这些结果支持了一种替代模型，即单体需要被重塑以使其在引发中具有活性。3.朝向弧菌特异性抗菌剂的产生：RctB，仅在弧菌家族中保守，并且似乎理想地适合于开发特异性针对弧菌的潜在药物。在霍乱的情况下，虽然口服补液治疗是主要的，但抗菌治疗有时成为强制性的，霍乱弧菌也不例外，对多种抗生素产生耐药性。通过对蛋白质的系统结构-功能分析，我们试图将注意力集中在对弧菌复制起始和生存至关重要的蛋白质区域。这些区域可以专门针对药物设计。4. Chr2的复制起始：复制起点链的打开。DNA上的所有交易都需要链打开，这是一种能量上不利的反应。细胞如何克服这种能量障碍进行复制启动尚不清楚。该假说是起始物与起始物结合在DNA上产生扭转应力，该扭转应力在邻近的AT富集区域中释放，该区域更容易熔化。然而，需要通过捕获至少一条单链来稳定熔融，否则应力将扩散出原点。在Chr2起源中已经获得了有利于稳定化的证据。我们发现Chr2起始子RctB具有单链DNA结合活性，该活性在体外通过特定的起源双链位点而大大稳定。稳定性源于引发剂与单链和双链位点形成的三元复合物。同时结合到两种类型的网站在起点似乎是一个共同的机制，细菌复制启动子稳定一个开放的起点。因此，与起始双链位点结合的引发剂通过有助于通过DNA应激引发开放和通过捕获单链稳定开放而发挥双重作用。