Characterization of the RRS: a new chromosomal structural element in E. coli

项目概要/摘要 细菌染色体是高度结构化的，以便将它们的大尺寸容纳在相对小的染色体中。 蜂窝式封装。在大肠在大肠杆菌中，染色体被分成31个染色体相互作用域（CID）， 几个较大的拓扑隔离的宏域。定义宏畴边界的结构是 未知约1百万bp的一个宏结构域包含复制末端，并被称为“复制区”。 Ter宏域。我们在大肠杆菌中发现了两个222 bp的基因间重复序列。大肠杆菌基因组， 对称地排列在复制末端周围，并且刚好在已被定义为Ter的外部。 宏域这些序列，现在被称为复制风险序列或RRS，触发不寻常的RecA水平 沉积在局部单链间隙中。RRS影响复制并且在肠杆菌中高度保守， 包括许多病原体。缺失一个RRS会产生生长缺陷。无法删除 这两个RRS，表明至少保留其中一个是必不可少的。RRS代表了一种新的基因组 这种现象并可能代表涉及基因组复制的染色体结构特征， 冷凝、分离或三者都有。我们推测RRS的功能是缓解拓扑压力。 RRS可以表示Ter宏域边界的物理现实。由于完全缺乏 关于RRS的信息，我们提出了一个一般的表征，以了解它们对复制的影响 和转录。所述方法包括多种标准遗传学、显微镜、细胞生物学、分子生物学、细胞生物学和细胞生物学。 生物学和基因组学。非标准方法包括一种新发明的基因组学方法， 进行了称为ssGAP-seq的基因组单链DNA探测和单分子复制测定 体外我们的目标是基本了解RRS在细菌核酸代谢中的作用，为将来的研究提供信息。 工作

Project Summary/Abstract Bacterial chromosomes are highly structured in order to accommodate their large size in a relatively small cellular package. In E. coli, the chromosome is divided into 31 chromosomal interaction domains (CIDs) and several larger and topologically isolated macrodomains. The structures defining macrodomain boundaries are unknown. One macrodomain of about 1 million bp encompasses the replication terminus and is referred to as the Ter macrodomain. We have discovered two 222 bp and intergenic repeat sequences in the E. coli genome, symmetrically arranged around the replication terminus and just outside what has been defined as the Ter macrodomain. These sequences, now called replication risk sequences or RRS, trigger unusual levels of RecA deposition in local single-stranded gaps. The RRS affect replication and are highly conserved in enterobacteria, including many pathogens. Deletion of one RRS generates a growth defect. It has not been possible to delete both RRS, suggesting that the retention of at least one of them is essential. The RRS represent a new genomic phenomenon and likely represent a chromosomal structural feature involved in genomic replication, condensation, segregation, or all three. We hypothesize that the function of RRS is to relieve topological stress. The RRS may represent the physical reality of the Ter macrodomain boundaries. Given a complete lack of information about RRS, we are proposing a general characterization to understand their effects on replication and transcription. The methods include a variety of standard genetics, microscopy, cell biology, molecular biology, and genomics. Nonstandard methods include a newly devised genomics method that allows the probing of genomic single-stranded DNA called ssGAP-seq and single molecule replication assays carried out in vitro. The goal is basic understanding of the role of RRS in bacterial nucleic acid metabolism to inform future work.