Mechanisms of Chromosome Maintenance in Bacteria

在霍乱弧菌的两条染色体中，较大的一条(ChR1)携带大部分看家基因，被认为是主染色体。较小的染色体(ChR2)似乎是从一个质粒进化而来的。虽然在细菌中普遍存在着染色体外的成分，但很少发现整合到染色体中并驱动染色体复制的质粒。一个原因可能是，质粒源的激发通常不限于细胞周期中的特定时间，而适时激发是生命所有领域中染色体起源的标准。因此，比较质粒和ChR2复制启动机制对于理解生物过程的时间如何从细胞周期中的随机进化为特定是有价值的。ChR2复制的时间取决于之前在ChR1中复制站点CRT。我们在2014年发现了这个位点，ChR2启动子RctB也在这里结合，这表明染色体确实可以积极沟通，并鼓励其他实验室研究两条染色体之间的复制协调机制。在真核生物中，来自不同来源的不协调复制会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之间用于复制启动的通讯：在细菌中鉴定新的检查点控制。CRT在ChR1上的位置是这样的，它会在ChR2复制开始之前复制。这为通信提供了一种直接的机制：ChR1复制首先启动。当叉子通过CRT时，它激活结合的RctB启动子分子，从而触发ChR2复制。因此，CRT的复制消除了阻止ChR2复制的检查点。然而，CRT激活ChR2复制的机制细节仍然是个谜。该活性似乎是RctB的重塑，它具体地改善了启动子的起始结合活性。正如预期的那样，启动子与起始点的结合是如此基本的要求，以至于当RctB随机突变但选择其启动子功能时，突变体仍然无一例外地对CRT做出反应。换句话说，CRT控制着RctB的一些基本功能，而这些功能是不能被灭活的。似乎起始点结合就是启动子的这样一种活动。去年，我们证明了CRTS的活性依赖于一个全球转录调节因子LRP，该调控因子也与CRT和RctB结合在一起。我们目前的研究表明，LRP激活RctB结合至少有两种机制：一种是通过LRP与CRT的直接蛋白质相互作用来帮助负载RctB；另一种是通过改变CRTS的DNA结构来明显增加RctB与CRTS DNA的亲和力。2.二聚反应对引发剂功能的控制。蛋白质的功能通常受齐聚作用的控制。RctB可以有效地进行二聚反应，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的二聚化结构域上进行了几个结构指导的突变。突变株存在不同程度的二聚化缺陷，且二聚化缺陷与启动子功能呈负相关。这表明二聚化是复制起始的一种抑制机制。这些突变体之所以获得回报，有两个原因：它们不再对CRT有反应，表明CRT可以通过二聚体解离激活RctB；它们对分子伴侣有反应，表明单体作用不足以解除伴侣需求。这些结果支持另一种模型，即需要对单体进行改造才能在引发过程中发挥活性。3.针对弧菌特异性抗菌剂的产生：RctB仅在弧菌家族中保守，似乎非常适合于开发针对弧菌的潜在药物。就霍乱而言，尽管口服补液治疗是主要的，但抗菌治疗有时也成为强制性的，霍乱弧菌也不例外，对多种抗生素产生抗药性。通过对该蛋白质进行系统的结构-功能分析，我们正试图将重点放在该蛋白质的一些区域，这些区域对复制启动和弧菌的生存至关重要。这些区域可以作为药物设计的特定目标。4.ChR2的复制启动：打开复制起始链。DNA上的所有交易都需要打开链，这在能量上是不利的反应。细胞如何克服这一能量障碍以启动复制尚不清楚。假设是，启动子与起始点的结合在DNA上产生扭转应力，在邻近的富含AT的区域释放，更容易熔化。然而，需要通过捕获至少一条单链来稳定熔化，否则应力将扩散到原点之外。在ChR2的起源中，已经获得了有利于稳定的证据。我们发现ChR2启动子RctB具有单链DNA结合活性，这种结合活性在体外被来源的特定双链位点极大地稳定。稳定性来源于引发剂与单链和双链中心形成的三元络合物。同时与起源地的两种位点结合似乎是细菌复制启动子稳定开放起始点的一种常见机制。因此，启动子与起始双链位点的结合具有双重作用，既有助于通过DNA胁迫启动开放，也有助于通过捕获单链来稳定开放。

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.