Chromosome Dynamics in Bacillus Subtills

描述(由申请人提供)：将复制的染色体压缩成形态和空间上不同的姐妹染色单体对于所有生物体中忠实的DNA分离是必不可少的。这是一个复杂的、鲜为人知的过程，受DNA的物理和机械性质以及特定蛋白质和酶的作用控制。在细菌中，染色体压缩只需要一小组基本因子，这为理解它们的作用原理提供了希望。 我们已经开发并调整了一套新的分子和细胞学分析方法，这将使我们能够解决模式生物枯草芽孢杆菌中DNA紧凑和组织的具体步骤。在这里，新复制的DNA由拓扑异构酶的组合活性压缩，拓扑异构酶产生相互缠绕的(超级螺旋)环，弯曲DNA的小核蛋白相关蛋白，以及被认为连接DNA片段的SMC凝集素复合体。我们知道，紧密排列的染色体的组织包括在短长度和长长度尺度上的折叠，但这两个层次的组织看起来是什么样子，它们是如何协调的还不清楚。利用全基因组的染色体构象捕获，辅以定量细胞学分析，我们将定义短长度尺度上的紧凑和长长度尺度上的染色体组织。这一新的描述水平将使我们能够确定压缩染色体的一小部分基本因素和打开DNA的转录机制之间的贡献和相互作用。特别是，我们的目标是深入了解高度保守的SMC凝集素复合体的作用，我们对此知之甚少。我们的初步数据表明，传统的遗传筛选遗漏了参与染色体紧凑和分离的重要因素，我们将采用新的细胞学方法和高通量合成致死筛选来鉴定它们。我们的具体目标是：1)确定SMC凝集素复合体对体内染色体构象的贡献；确定PARB/PARS和高转录基因在SMC介导的起源分离中的作用；以及SMC是如何在这些座位上富含的。2)使用全基因组的相互作用频率，定义DNA如何在整个染色体的短长度尺度上被压缩。3)使用高通量细胞学和合成致死筛选来鉴定新的染色体组织和分离因素。

DESCRIPTION (provided by applicant): Compaction of replicated chromosomes into morphologically and spatially distinct sister chromatids is essential for faithful DNA segregation in all organisms. This is a complex, poorly understood process that is controlled by the physical and mechanical properties of DNA as well as the action of specific proteins and enzymes. In bacteria, only a small set of essential factors are required for chromosome compaction, offering the hope that it will be relatively straightforward to understand the principles by which they act. We have developed and adapted a new set of molecular and cytological assays that will allow us to address specific steps in DNA compaction and organization in the model organism Bacillus subtilis. Here, newly replicated DNA is compacted by the combined activities of topoisomerases that generate interwound (supercoiled) loops, small nucleoid-associated proteins that bend DNA, and SMC condensin complexes, which are thought to bridge DNA segments. We know that the organization of the compacted chromosome involves folding both at short and long length-scales, but what these two levels of organization look like and how they are coordinated are unclear. Using genome-wide chromosome conformation capture complemented by quantitative cytological assay, we will define compaction on short length-scales and chromosome organization on long length-scales. This new level of description will then allow us to establish the contributions of and interplay between the small set of essential factors that compact the chromosome and the transcription machinery that opens up the DNA. In particular, we aim to gain insight into the role of the highly conserved SMC condensin complex, for which we have the least information. Our preliminary data indicate that important factors that participate in chromosome compaction and segregation have been missed by traditional genetic screens, and we will take a novel cytological approach and high throughput synthetic lethal screens to identify them. Our specific aims are to: 1) Determine how the SMC condensin complex contributes to chromosome conformation in vivo; define the role of ParB/parS and highly transcribed genes in SMC-mediated origin segregation; and establish how SMC is enriched at these loci. 2) Define how DNA is compacted on short length-scales throughout the chromosome using genome-wide interaction frequencies. 3) Identify and characterize new chromosome organization and segregation factors using high throughput cytological and synthetic lethal screens.