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

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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.