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。质粒虽然在细菌中普遍存在，但很少作为驱动染色体复制的复制子的来源。一个原因可能是质粒起源的激活通常不局限于细胞周期的特定时间，而及时的激活在染色体起源中是常见的。因此，质粒和chr2系统的比较可能对理解细胞周期中生物过程的时间是如何调节的有价值。Chr2复制似乎也依赖于chrI复制。两条染色体如何沟通以协调复制在很大程度上是未知的，这些知识是我们理解细菌中多部基因组如何维持的基础。在真核生物中，除了不完全复制和过度复制外，不协调复制还会导致发育异常和癌症。最后，chr2的复制是由分离蛋白控制的。分离对复制影响的发现是细菌染色体动力学领域的最新进展，最近在酵母和人类细胞中也证实了复制和分离之间的串扰。我们在了解这些过程方面的进展报告如下。从随机到细胞周期调节复制起始的转变：chr2复制的初始特征表明，其控制将比其假定的祖质粒更为复杂。我们过去的研究发现了chr2起源的三个新特征：1)启动子结合位点的甲基化要求启动子结合。一些细菌起源有甲基化位点，起源的及时激活取决于甲基化；2)在起始点存在一种新的用于抑制复制的启动物结合位点（39-mers）；3)存在一个额外的调节反馈回路，以更严格地控制引发剂的合成。2013年，我们发现了另外三个控制chr2复制的调节因子，我们正在继续对其进行表征。因此，细胞周期对复制起始的调节似乎比调节质粒复制的机制更为复杂。下文将讨论新监管机构的作用机制。启动物二聚体参与chr2复制的控制：chr2启动物不具有任何酶活性，但通过二聚体调节其活性。只有单体与复制起始点结合才会导致起始。二聚体与单体竞争结合原点，从而阻碍起始。二聚化还排除了引发剂与复制抑制39-mer的结合，39-mer仅结合单体。因此二聚化也有助于起始。因此，控制启动器的关联状态对于控制chr2复制至关重要。我们已经确定了涉及起源和39-mer DNA结合和二聚化的引发剂结构域。这三种活性的重要残基位于658个残基长蛋白中的71个。这三个活动的领域也重叠，解释了一个领域的变化如何影响其他两个领域的活动。二聚化和DNA结合之间的相互作用在自然界是众所周知的，但它似乎在控制chr2复制中起着更深远的作用。chr2复制由chrI中的一个位点控制：我们假设霍乱弧菌的两条染色体的及时复制和分离需要它们之间的通信，因此它们都可以在细胞分裂之前完成这一过程。染色体间通讯的初步证据已经获得。我们在chr1上发现了一个位点，可能是通过重塑chri的启动子来显著刺激chr2的复制。DNA位点也使得正常的伴侣蛋白（DnaK）依赖的chr2伴侣蛋白不依赖的复制。对于许多DNA结合蛋白，已知特异性结合可以重塑蛋白质。chrI位点的新颖之处在于它的序列与已知的启动物的特定结合位点没有相似性。表征这种非典型dna -蛋白质相互作用是我们近期的目标。无论如何，这一发现表明了一种协调两条染色体复制的机制。分离蛋白对chr2复制的控制：分离影响复制的事实直到最近才为人所知。我们对细菌中染色体分离的认识主要来自对质粒的研究，在质粒中首次发现了专门用于分离的基因（par基因）。质粒par基因的同源物现已在大多数细菌（包括霍乱弧菌）的复制起源附近被鉴定出来。弧菌的两条染色体都有自己的par基因。我们之前已经证明，chr1的一个par基因（parA1）特异性地促进了chr1的复制。在枯草芽孢杆菌中也有类似的发现。在枯草芽孢杆菌和霍乱弧菌中，普遍的细菌引发剂dna被发现是Par蛋白的直接靶点。我们现在发现chr2的ParB2也可以特异性地刺激chr2的复制，但通过不同的机制。ParB2可以扩散到着丝粒之外，在那里它们通常结合到邻近的复制起点，并覆盖一个复制抑制39-mer位点。这可能会干扰39-mer与引发剂的相互作用，这是其复制抑制功能所必需的。ParB2也可以通过直接结合不同的39-mer来促进复制，而不需要着丝粒。因此，ParB2似乎通过与两个强复制抑制位点的结合竞争来促进复制，通过扩散到一个位点并直接结合到另一个位点。扩散代表了一种新的远程复制控制机制，而非着丝粒结合是一种由分离蛋白控制的新机制。这些研究确立了分离蛋白影响复制的几种途径。弧菌特异性抗菌剂的产生：鉴于多重耐药致病性弧菌的日益流行，需要新的靶点和药物来对抗这些病原体。chr2启动子RctB仅在弧菌科中保守，似乎非常适合开发潜在的抗弧菌特异性药物。靶蛋白的三维结构信息为药物设计提供了极大的便利。为了实现这个目标，我们已经开始对RctB进行系统的域分析。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，需要伴侣蛋白重塑和/或与特定DNA结合才能产生活性。在与Alex Wlodawer （CCR）的合作中，虽然最初试图形成RctB晶体的尝试失败了，但我们正在尝试通过缺失分析来分离功能域，希望更小的功能区域可能更适合结晶。我们还试图通过部分蛋白水解来识别结构域，并通过MassSpec来识别产品（与CCR的Lisa Jenkins合作）。这些较小的衍生品可能更适合于结构研究。我们特别乐观地认为，上述71个氨基酸长片段的结构测定将有可能通过核磁共振（与白亚文，CCR合作），因为它的小尺寸。这个小区域可能是一个有吸引力的药物靶标，因为它包含三个重要的域的引发剂。

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 in bacteria, rarely serve as the source of replicons that drive chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time of the cell cycle, whereas timely firing is common among chromosomal origins. Comparison of plasmid and chr2 systems could thus be valuable for understanding how the timing of a biological process is regulated in the cell cycle. Chr2 replication seems also to depend on chrI replication. How the two chromosomes communicate to coordinate replication is largely unknown and this knowledge is basic to our understanding of how multipartite genomes are maintained in bacteria. In eukaryotes, in addition to incomplete and over-replication, uncoordinated replication causes developmental abnormalities and cancer. Finally, chr2 replication is controlled by a segregation protein. The discovery of the influence of segregation on replication is a recent development in the field of chromosome dynamics in bacteria, and cross-talk between replication and segregation has been demonstrated recently in yeast and human cells. Our progress in understanding these processes is reported below. Transition from random to cell-cycle regulated replication initiation: The initial characterization of chr2 replication suggested that its control would be more complex than that of its presumed progenitor plasmid. Our past studies identified three new features of the chr2 origin: 1) Requirement for methylation of initiator binding sites in the origin for initiator binding. Several bacterial origins have methylation sites and timely firing of origins depends on methylation; 2) Presence of a new kind of initiator binding site (39-mers) in the origin exclusively for inhibiting replication; and 3) Presence of an extra regulatory feedback loop to control initiator synthesis more tightly. In 2013, we discovered three additional regulators in the control of chr2 replication, which we are continuing to characterize. The cell-cycle regulation of replication initiation thus appears to be considerably more involved than the mechanisms that regulate plasmid replication. The mechanisms of action of the new regulators are discussed below. Participation of initiator dimers in the control of chr2 replication: The chr2 initiator does not have any enzymatic activity but uses homodimerization to regulate its activity. Only monomer binding to the replication origin leads to initiation. The dimers compete with monomers for binding to the origin and thus hinders initiation. The dimerization also precludes initiator binding to replication inhibitory 39-mer, which binds only the monomers. The dimerization thus can help initiation also. Controlling the association state of the initiator is thus critical for controlling chr2 replication. We have determined the domains of the initiator involved in origin and 39-mer DNA binding and in dimerization. Important residues for all three activities are found to reside within 71 out of the 658 residue long protein. The domains for the three activities also overlap explaining how changes in one domain can influence the activities of the other two domains. The interplay between dimerization and DNA binding is well-known in nature but it seems to play a more profound role in controlling chr2 replication. Control of chr2 replication by a site in chrI: We hypothesize that the timely replication and segregation of the two chromosomes of V. cholerae would require communication between them, so that both can complete the processes prior to cell division. Preliminary evidence for inter-chromosomal communication has been obtained. We identified a locus on chr1 that can significantly stimulate chr2 replication, likely by remodeling the chrII initiator. The DNA site also made the normally chaperone (DnaK)-dependent replication of chr2 chaperone-independent. For many DNA binding proteins, specific binding is known to remodel the protein. The novelty of the chrI site is that its sequence has no similarity to the known specific binding sites of the initiator. Characterization of this atypical DNA-protein interaction is our goal for the immediate future. In any event, the finding suggests a mechanism that may coordinate replication of the two chromosomes. Control of chr2 replication by a segregation protein: The fact that segregation can influence replication was not known until recently. Our knowledge of chromosome segregation in bacteria comes primarily from studies of plasmids, where genes dedicated to segregation (par genes) were first found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both of the Vibrio chromosomes have their own par genes. We showed earlier that one of the par genes (parA1) of chr1 specifically promoted chr1 replication. A similar finding was also made in B. subtilis. In both B. subtilis and V. cholerae, the universal bacterial initiator, DnaA, was found to be the direct target of Par proteins. We now find that ParB2 of chr2 can also specifically stimulate replication of chr2 but through different mechanisms. ParB2 can spread beyond the centromere where they normally bind into the adjacent replication origin and cover one of the replication inhibitory 39-mer sites. This likely interferes with the 39-mer interaction with the initiator, which is required for its replication inhibitory function. ParB2 could also promote replication without requiring the centromere by directly binding to a different 39-mer. ParB2 thus appears to promote replication by competing with the initiator for binding to two strong replication inhibitory sites, by spreading into one and directly binding to the other. Spreading represents a novel replication control mechanism that acts from a distance, whereas non-centromeric binding is a novel mechanism for control by a segregation protein. These studies establish several ways by which segregation proteins could influence replication. Generation of Vibrio-specific antimicrobial agents: Given the increasing prevalence of multi-drug resistant pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. The chr2 initiator, RctB, is conserved only in the family Vibrionaceae and appears ideally suited for developing potential anti-vibrio specific drugs. 3-D structural information of target proteins greatly facilitates drug design. Towards this goal we have started a systematic domain analysis of RctB. Replication initiators in general have proven refractory to structural studies most likely because they have unstructured regions and require remodeling by chaperone proteins and/or binding to specific DNA for activity. In collaboration with Alex Wlodawer (CCR), although initial attempts to form crystals of RctB have failed, we are attempting deletion analysis to isolate functional domains, hoping that smaller functional regions might be better suited for crystallization. We are also trying to identify domains by partial proteolysis and identity the products by MassSpec (in collaboration with Lisa Jenkins, CCR). These smaller derivatives might be more amenable to structural studies. We are particularly optimistic that structure determination of the 71 amino acids long fragment described above will be possible by NMR (in collaboration with Yawen Bai, CCR) because of its small size. This small region could be an attractive drug target as it contains three important domains of the initiator.