Chromosome Dynamics in Bacillus Subtills

描述（由申请人提供）：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的DNA分离是必不可少的。这是一个复杂的、知之甚少的过程，它受DNA的物理和机械特性以及特定蛋白质和酶的作用控制。在细菌中，染色体致密化只需要一小部分基本因子，这为理解它们的作用原理提供了希望。 我们已经开发和适应了一套新的分子和细胞学检测，这将使我们能够解决模式生物枯草芽孢杆菌中DNA压缩和组织的具体步骤。在这里，新复制的DNA被拓扑异构酶的组合活动压缩，拓扑异构酶产生相互缠绕（超螺旋）的环，小的核苷酸相关蛋白质，弯曲DNA，和SMC凝聚素复合物，被认为是桥接DNA片段。我们知道，紧密染色体的组织包括短尺度和长尺度的折叠，但这两个层次的组织是什么样子的，以及它们是如何协调的，还不清楚。使用全基因组染色体构象捕获，辅以定量细胞学分析，我们将定义压缩短长度尺度和染色体组织的长度尺度。这一新的描述水平将使我们能够建立一小部分压缩染色体的基本因子和打开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凝聚复合物的作用，我们有最少的信息。我们的初步数据表明，参与染色体压缩和分离的重要因素已被传统的遗传筛选所遗漏，我们将采取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凝聚素复合物如何有助于体内染色体构象;确定ParB/parS和高度转录的基因在SMC介导的起源分离中的作用;并确定SMC如何在这些位点富集。2)定义DNA是如何压缩在短长度尺度上整个染色体使用全基因组的相互作用频率。3)使用高通量细胞学和合成致死筛选鉴定和表征新的染色体组织和分离因子。

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.