Mechanisms of Chromosome Maintenance in Bacteria

在两条霍乱弧菌染色体中，较大的一条（Chr1）携带着大部分的管家基因，被认为是主要染色体。较小的染色体（Chr2）似乎是从质粒进化而来的。质粒虽然在细菌中作为染色体外元件普遍存在，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激活通常不局限于细胞周期中的特定时间，而及时激活是生命所有领域中染色体起源的常态。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如何在细胞周期中从随机演变为特异性是有价值的。Chr2复制的时间取决于Chr1中某个位点先前的复制。我们在2014年发现了这个位点，这表明染色体之间确实存在交流，并鼓励了其他实验室对两条染色体之间复制协调机制的研究。在真核生物中，来自不同起源的不协调复制会导致发育异常和癌症。我们在了解Chr2的复制及其与Chr1的协调方面的进展如下。1. Chr2的复制起始：Chr2复制的初始特征表明，其调控比其假定的祖质粒更为复杂。许多（如果不是全部的话）这些调控是通过Chr2特异性启动蛋白RctB介导的。我们已经开始对该蛋白进行系统的结构-功能分析，不仅是为了更深入地了解Chr2复制控制，也是为了设计抗霍乱弧菌的药物（下文第2节）。在过去的一年里，我们成功地鉴定了一个与分子伴侣dna （Hsp70）相互作用的RctB新结构域。DnaK增加了RctB与Chr2复制起点的结合，这一观察结果足以解释为什么DnaK是Chr2复制所必需的。DnaK也帮助RctB结合到复制起点以外的区域以达到调控的目的。RctB的DNA相互作用域（K-domain）的鉴定为DnaK如何帮助控制RctB的两种不同的DNA结合活性提供了有价值的见解。k结构域的突变使RctB的复制启动子活性失活，而允许启动子活性的基因内抑制子似乎是通过降低蛋白质的复制抑制活性来实现的。这些结果在确定Chr2复制的主要负调控机制方面是有益的。目前，我们正试图确定RctB结构域与DnaK共同伴侣DnaJ的相互作用。2. 针对弧菌特异性抗菌剂的产生：Chr2引发剂RctB仅在弧菌家族中保守，似乎非常适合开发针对弧菌的潜在药物。就霍乱而言，虽然口服补液治疗是主要手段，但有时必须进行抗微生物治疗，霍乱弧菌对多种抗生素产生耐药性也不例外。当目标蛋白的三维结构信息可用时，极大地促进了新药的设计。一般来说，复制启动子已被证明难以进行结构研究，这很可能是因为它们具有非结构化区域，并且需要伴侣蛋白重塑才能发挥活性，这似乎是RctB的情况。在与Alex Wlodawer (CCR), Lisa Jenkins （CCR）和Rodolfo Ghirlando （NIDDK）的合作下，我们在最近几个月成功地解决了一半RctB的结构。许多调控突变被定位在这个区域。迄今为止，确定具有基本功能的其余区域的结构已被证明是困难的，可能是因为这些区域本质上是非结构化的（来自与CCR的Yawen Bai合作获得的核磁共振证据）。这些区域的折叠可能需要它们相互作用的伙伴的存在。目前，我们正准备在Frederick的NCI核心设施尝试cryo-EM来解决RctB及其合作DNA结合位点的结构。Chr1和Chr2之间的复制协调：鉴定一种新的细菌检查点控制。我们假设两条霍乱弧菌染色体的及时复制和分离需要它们之间的通信，因此这两个过程都在细胞分裂之前完成。当我们发现Chr1上的一个位点（crtS为Chr2复制触发位点）可以结合RctB并显著刺激Chr2复制时，获得了染色体间通信的证据。crtS在Chr1上的位置决定了它会在Chr2复制开始之前进行复制。这为通信提供了一种直接的机制。首先启动Chr1复制。当叉穿过crtS时，它激活绑定的Chr2启动器，触发Chr2复制。因此，crtS的复制解除了阻止Chr2复制的检查点。我们现在已经证明，阻断Chr1复制也会阻断Chr2复制，证明Chr2复制确实依赖于Chr1复制。引人注目的是，两个未复制的crtS副本也可以允许Chr2复制，这表明crtS在没有自身复制的情况下具有显著的活性，复制的作用是通过加倍crtS基因剂量来增加这种活性。这种活性似乎是重塑RctB，因为crtS可以减轻引发剂对分子伴侣的依赖。该研究提供了一个新的例子，如何加倍基因剂量的复制是用于其他调控目的。目前，我们正试图了解dna -蛋白质在crtS中的相互作用是如何重塑RctB的。crtS位点最小长度约为70 bp，这对于蛋白质结合位点来说太大了。我们在2018年发现，一种保守的全局转录因子Lrp（亮氨酸反应蛋白）也与crtS结合，并显著刺激RctB同时与crtS结合，这部分解开了这个谜团。crtS如何刺激RctB结合，以及这种结合如何重塑RctB以激活其启动器功能是我们目前正在解决的问题。4. 2018年，我们开始着手解决一个古老但尚未解决的问题，即DNA链是如何打开的。DNA上的所有交易都需要打开链，这是一种能量上不利的反应。细胞如何克服这种能量屏障还不清楚。我们关注的是复制起始链是如何打开的。假设是，与原点结合的引发剂在DNA上产生扭转应力，这种应力被释放到邻近的富含at的区域，这更容易融化。然而，需要通过捕获至少一条单链来稳定熔化，否则应力会从原点扩散出去。支持稳定的证据已经获得。5. 在与谢菲尔德大学（英国）的Ling Chin Hwang的合作中，我们正试图了解Chr2是如何使用两种蛋白质ParA和ParB分离的。这是一个合作，我们创造菌株，做一些细胞生物学而凌做生物化学和生物物理学。这里的挑战是理解姐妹染色体如何在没有纺锤体或“有丝分裂”马达存在的情况下以定向方式运动。

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 in Chr1. Our discovery of this site (in 2014) demonstrated that chromosomes do 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. Replication initiation of Chr2: The initial characterization of Chr2 replication suggested that its regulation is more complex than that of its presumed progenitor plasmid. Many, if not all, of these regulations are mediated through the Chr2 specific initiator protein, RctB. We have embarked on a systematic structure-function analysis of the protein not only to understand Chr2 replication control in greater depth but also for drug design against V. cholerae (Section 2 below). In the past year, we succeeded in identifying a new domain of RctB that interacts with the molecular chaperone DnaK (Hsp70). DnaK increases RctB binding to the replication origin of Chr2 and this observation suffices to explain why DnaK is required for Chr2 replication. DnaK also helps RctB binding to regions outside of the replication origin for regulatory purposes. Identification of the DnaK interacting domain of RctB (K-domain) provided valuable insights on how DnaK helps in controlling the two different kinds of DNA binding activity of RctB. Mutations in the K-domain inactivates the replication initiator activity of RctB and intragenic suppressors that allow initiator activity seem to have done so by reducing replication inhibitory activities of the protein. These results have been rewarding in identifying the major negative regulatory mechanisms of Chr2 replication. At present, we are trying to identify the RctB domain for interaction with the DnaK co-chaperone, DnaJ. 2. Towards generation of Vibrio-specific antimicrobial agents: The Chr2 initiator, 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. Design of new drugs is greatly facilitated when the 3-D structural information of target proteins are available. Replication initiators in general have proven refractory to structural studies most likely because they have unstructured regions and require remodeling by chaperone proteins for activity, which seems to be the case for RctB. In collaboration with Alex Wlodawer (CCR), Lisa Jenkins (CCR) and Rodolfo Ghirlando (NIDDK), we succeeded in recent months solving the structure of one half of RctB. Many of the regulatory mutations are mapped in this region. Determination of the structure of the remaining regions that serve essential functions has so far proven refractory, possibly because such regions could be intrinsically unstructured (from NMR evidence obtained in collaboration with Yawen Bai, CCR). The folding of these regions may require the presence of their interacting partners. At present we are gearing up to try cryo-EM to solve the structure of RctB with its partner DNA binding site in the NCI core facility in Frederick. 3. Replication coordination between Chr1 and Chr2: Identification of a novel check point control in bacteria. We hypothesized that the timely replication and segregation of the two V. cholerae chromosomes would require communication between them, so that both the processes are completed prior to cell division. An evidence for inter-chromosomal communication was obtained when we identified a site on Chr1 (crtS for Chr2 replication triggering site) that can bind RctB and significantly stimulate Chr2 replication. 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 Chr2 initiator that triggers Chr2 replication. Replication of crtS thus relieves the check point that prevents Chr2 replication. We have now shown that blocking of Chr1 replication also blocks Chr2 replication, demonstrating that Chr2 replication indeed depends on Chr1 replication. Strikingly, two unreplicated copies of crtS could also allow Chr2 replication, suggesting that crtS has significant activity without itself being replicated and a role of replication is to increase this activity by doubling the crtS gene dosage. The activity appears to be remodeling of RctB, since crtS could relieve the dependence of the initiator on molecular chaperones. The study provides a novel example of how doubling of gene dosage by replication is utilized for other regulatory purposes. At present, we are trying to understand how DNA-protein interactions at crtS remodels RctB. crtS site is minimally about 70 bp long, which is too large for a protein binding site. The part of this mystery is solved by our discovery in 2018 that a well conserved global transcription factor Lrp (Leucine responsive protein) also binds to crtS and significantly stimulates simultaneous RctB binding to crtS. How crtS stimulates RctB binding and how the binding remodels RctB to activate its initiator function are the questions that we are currently addressing. 4. In 2018, we initiated addressing an age-old yet unsolved question on how opening of the strands of DNA comes about. All transactions on DNA require strand-opening which is an energetically unfavorable reaction. How cells overcome this energy barrier is not clearly understood. We are focussing on how strands of replication origin opens. 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. 5. In collaboration with Ling Chin Hwang of Sheffield U (UK), we are trying to understand how Chr2 segregates using two proteins, ParA and ParB. This is a collaboration where we create strains and do some cell biology whereas Ling does the biochemistry and biophysics. The challenge here is to understand how sister chromosomes move in a directed fashion without the presence of spindle or "mitotic" motors.