Mechanisms of Chromosome Maintenance in Bacteria

细菌染色体维持机制

• 批准号: 7592644
• 负责人: DHRUBA K CHATTORAJ
• 金额: $ 99.48万
• 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/7592644
• 关键词: Actins; Aneuploidy; Antibodies; Bacteria; Bacterial Chromosomes; Bacterial Infections; Bacterial Model; Behavior; Binding; Binding Sites; Cell Cycle; Cell Extracts; Cells; Centromere; Characteristics; Chromosome Segregation; Chromosomes; Communication; Complex; Condition; Control Locus; Controlled Study; DNA; DNA biosynthesis; Development; Disease; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Frequencies; Gene Expression; Genes; Genome Stability; Growth; Growth and Development function; Homologous Gene; Immunoblotting; Localized; Maintenance; Microtubules; Mitotic; Mitotic Spindle Apparatus; Morphology; Motor; Numbers; Organism; Plasmids; Play; Property; Proteins; Rate; Regulation; Relative (related person); Replication Initiation; Replication Origin; Role; Sister; Site; Site-Specific DNA-Methyltransferase (Adenine-Specific); Structure; System; Testing; Therapeutic; Time; Vibrio; Vibrio cholerae; cancer cell; cell growth; chromosome replication; interest; melting; novel; promoter; repository; segregation

A recent development in the field of E. coli DNA replication is the identification of weak (incognito) binding sites for the initiator DnaA in the origin of replication, called I-sites. These sites play a key role in timing of replication initiation in the cell cycle. Because of the difficulty of recognizing I-sites, the generality of their occurrence has remained obscure. Richard Fekete and Tatiana Venkova-Canova have succeeded in localizing sequences with characteristics of an I-site in a second system (in the replication origin of plasmid P1). The sequence information should help to define an I-site better and thus help identification of such sites in other systems and study their roles in replication control. Bacteria capable of rapid proliferation such as E. coli and B. subtilis usually amplify the origin proximal region of their chromosome. In V. cholerae, Preeti Srivastava found this to be true for chromosome I but not II. The growth-rate insensitive behavior of the latter turned out to be due to homeostatic controls on its initiator RctB. Relaxing the control increased the copy number of chromosome II but decreased the cell growth rate, suggesting that this chromosome might serve as a repository for potentially deleterious genes. These genes thus could play a role in maintaining relative copy number of the two chromosomes. Ryosuke Kadoya is standardizing conditions to selectively inhibit replication initiation of one of the two Vibrio chromosomes and study the consequences to replication and segregation of the other chromosome, as well as to cell growth and division. These studies are expected to provide initial evidence for communication between the two chromosomes and whether mechanisms exist in bacteria analogous to check-point controls prevalent in eukaryotes. To study the regulation of replication of chromosome II, Tatiana Venkova-Canova has defined the bounds of the replication origin and adjoining sequences that serve to control replication initiation negatively. The origin resembles those of some plasmids but the negative control locus is more extended and complex compared to the plasmids. A novel mechanism was found that controls the level of chromosome II specific initiator, RctB, homeostatically, and thereby the replication initiation frequency. Unlike in E. coli, where DNA adenine methylase (Dam) plays a facilitatory role in DNA replication, the enzyme appears essential for replication of both the chromosomes of V. cholerae. The exact role of Dam in replication is not known but most likely it alters the structure of origin DNA that eases its melting, a crucial step in replication initiation. Dam can also modulate gene expression by methylating promoter DNA, and could play an indirect role by supplying a gene product crucial for replication initiation. Gaelle Demarre is testing these alternate possibilities on how Dam could be playing its obligatory role in V. cholerae. The equivalent of microtubules of the eukaryotic mitotic apparatus has not been found in bacteria, and how the bacterial chromosomes segregate has remained largely enigmatic. The recent discovery in bacteria of an actin homolog, MreB, is of special interest because of its suggested role in chromosome segregation. Preeti Srivastava found that the properties of V. cholerae MreB to be similar to its homolog in other bacteria, and that alteration of the protein led to gross distortion in nucleoid morphology and localization of centromeric regions for both the chromosomes. In V. cholerae, Ranajit Ghosh has identified the centromere for chromosome I. A couple of centromere-associated proteins (ParA and ParB) have been purified. To understand how the centromere is utilized to segregate and retain the sister chromosomes in opposite cell halves, Ranajit Ghosh is trying to identify proteins that may serve as mitotic motors or chromosomal anchors. He has been able to co-immuno-precipitate several proteins from crude cell extracts using antibodies against ParAB proteins. One of the proteins has been identified to be MreB by immunoblotting, and the others are being identified using LCMS/MS

在大肠杆菌DNA复制领域的一个最新进展是鉴定了复制起点上的启动子DNAA的弱(隐名)结合位点，称为I-位点。这些位点在细胞周期中复制起始的时间上起着关键作用。由于很难识别I-位点，它们出现的一般性仍然不清楚。Richard Fekete和Tatiana Venkova-Canova已经成功地将具有I-位特征的序列定位在第二系统中(在质粒P1的复制起始处)。序列信息应该有助于更好地定义I-位点，从而有助于识别其他系统中的此类位点，并研究它们在复制控制中的作用。能够快速增殖的细菌，如大肠杆菌和枯草杆菌，通常会扩增其染色体的起始近端区域。在霍乱弧菌中，Preeti Sriastava发现这一点适用于I号染色体，但不适用于II号染色体。后者的生长速度不敏感行为被证明是由于其启动子RctB的动态平衡控制。放松控制增加了第二号染色体的拷贝数，但降低了细胞生长速度，这表明该染色体可能是潜在有害基因的储存库。因此，这些基因可能在维持两条染色体的相对拷贝数方面发挥作用。Ryosuke Kadoya正在标准化条件，以选择性地抑制两条弧菌染色体之一的复制启动，并研究对另一条染色体复制和分离以及对细胞生长和分裂的影响。这些研究有望为两条染色体之间的交流以及细菌中是否存在类似于真核生物中普遍存在的检查点控制的机制提供初步证据。为了研究II号染色体的复制调控，Tatiana Venkova-Canova定义了复制起点和相邻序列的界限，这些序列用于负向控制复制起始。它的起源与一些质粒相似，但与质粒相比，负控制点的范围更广、更复杂。发现了一种新的机制，它以稳态的方式控制第二染色体特异性启动子RctB的水平，从而控制复制启动频率。在大肠杆菌中，DNA腺嘌呤甲基酶(DAM)在DNA复制中起促进作用，与之不同，该酶似乎对霍乱弧菌的两条染色体的复制都是必不可少的。Dam在复制中的确切作用尚不清楚，但最有可能的是它改变了来源DNA的结构，使其易于融化，这是复制启动的关键一步。DAM还可以通过甲基化启动子DNA来调节基因的表达，并通过提供对复制启动至关重要的基因产物来发挥间接作用。Gaelle DeMarre正在测试这些替代可能性，看看Dam如何在霍乱弧菌中发挥其必备作用。在细菌中还没有发现相当于真核有丝分裂装置的微管，细菌染色体是如何分离的在很大程度上仍然是个谜。最近在细菌中发现的肌动蛋白同源物MreB特别令人感兴趣，因为它被认为在染色体分离中起到了作用。Preeti Sriastava发现霍乱弧菌MreB的性质与其在其他细菌中的同源物相似，蛋白质的改变导致两条染色体的类核形态和着丝粒区域的严重扭曲。在霍乱弧菌中，Ranajit Ghosh已经确定了第一染色体的着丝粒，并纯化了几个着丝粒相关蛋白(ParA和PARB)。为了了解着丝粒是如何被用来分离和保留相对细胞一半中的姐妹染色体的，Ranajit Ghosh正在试图识别可能作为有丝分裂马达或染色体锚的蛋白质。他已经能够使用针对Parab蛋白质的抗体从粗细胞提取液中免疫共沉淀几种蛋白质。其中一个蛋白已经通过免疫印迹鉴定为MreB，其他蛋白正在用LC MS/MS鉴定