Mechanisms of Chromosome Maintenance in Bacteria

细菌染色体维持机制

• 批准号: 7338296
• 负责人: DHRUBA K CHATTORAJ
• 金额: --
• 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/7338296
• 关键词: Mechanisms; Chromosome; Maintenance; Bacteria

In the past year, we have brought to closure projects on replication control of a low copy number E. coli plasmid, P1. Nilangshu Das with help from a mathematician, Johan Paulsson (Cambridge U., UK), has characterized control-defective initiator mutants that confer relatively unhindered replication profiency to the plasmid. The properties of the mutants are best explained assuming multiple modes of control involving transcriptional autorepression of the initiator gene, initiator inactivation by dimerization, and origin inactivation by pairing. The control is best when the three mechanisms cooperate. A new development in the field of E. coli DNA replication is the finding of weak (incognito) binding sites for the initiator DnaA in the origin of replication, called I-sites. These sites recognize only by ATP-bound DnaA. The generality of I-sites in DNA replication remained to be established. Richard Fekete has identified a possible I-site in the replication origin in plasmid P1. Since ATP-DnaA level decreases after the initiation of chromosomal replication, this may be a way for the plasmid to correlate its replication with that of the chromosome and add a new paradigm in plasmid replication control.Knowing the importance for controlling initiator proteins in DNA replication control, Debasish Pal has studied the regulation of the RctB initiator for chromosome II replication of V. cholerae. A strong promoter for the gene was identified and found to be autorepressed as well as regulated by unstream sequences and global regulators, IHF and Dam methylase. Increasing RctB in trans increased the copy number of a miniplasmid carrying the chrII origin, implying that RctB can be rate-limiting for chrII replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis. To study the regulation of replication of chrII, Tatiana Venkova-Canova has defined the origin and its negative control elements. The origin resembles those of P1 type plasmids but the negative control locus is more extended and complex. It is already clear that the regulation of replication has diverged significantly from the ones operating in plasmids or in E. coli.Preeti Srivastava is studying chromosome dynamics in V. cholera using fluorescence microscopy and flow cytometry. So far she has tracked the movement of the origins and terminii of both the chromosomes. The migration patterns seem to differ between the two chromosomes as well as from what has been reported in other systems. One of the novel findings is the cohesion of the terminus region of the two chromosomes, which suggests the possibility that there could be segregational information in those regions.

在过去的一年里，我们已经结束了低拷贝数大肠杆菌质粒P1的复制控制项目。Nilangshu Das在数学家Johan Paulsson(英国剑桥大学)的帮助下，对控制缺陷的启动子突变进行了表征，这些突变赋予了质粒相对不受阻碍的复制倾向。可以很好地解释突变体的性质，假设有多种控制模式，包括转录自抑制启动子基因，通过二聚化使启动子失活，通过配对使起始子失活。当三种机制协同工作时，控制效果最好。大肠杆菌DNA复制领域的一项新进展是在复制起点发现了启动子DNAA的弱(隐蔽)结合位点，称为I-位点。这些位点只能通过三磷酸腺苷结合的DNAA识别。I位点在DNA复制中的共性尚待确定。Richard Fekete已经确定了在质粒P1的复制起始处可能存在一个I-位点。由于在染色体复制开始后，ATP-Dna A水平下降，这可能是一种将其复制与染色体复制联系起来的方式，并为质粒复制控制增加了一种新的范式。Debasish Pal认识到控制启动子蛋白在DNA复制控制中的重要性，研究了RctB启动子对霍乱弧菌第二染色体复制的调控。该基因的一个强大的启动子被鉴定出来，并被发现受到非序列和全局调控因子IHF和Dam甲基酶的自我抑制和调控。在反式中增加RctB可以增加携带chrII起始点的微型质粒的拷贝数，这意味着RctB可以限制chrII的复制。RctB上的多种控制模式有望减少启动子浓度的波动，从而有助于维持染色体拷贝数的动态平衡。为了研究chrII的复制调控，Tatiana Venkova-Canova定义了chrII的来源及其负控元件。其来源与P1型质粒区相似，但负控制区更为广泛和复杂。已经很清楚的是，复制的调控已经明显不同于在质粒或大肠杆菌中操作的调控。Preeti Sriastava正在使用荧光显微镜和流式细胞术研究霍乱弧菌的染色体动力学。到目前为止，她已经追踪到了这两条染色体的起点和终点的运动。这两条染色体之间的迁移模式似乎不同，也不同于其他系统中所报道的。其中一个新发现是两条染色体末端区域的凝聚力，这表明在这些区域可能存在分离信息。