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

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $198,828

## Abstract

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.

## Key facts

- **NIH application ID:** 10876389
- **Project number:** 5R21AI179102-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Michael M. Cox
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $198,828
- **Award type:** 5
- **Project period:** 2023-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10876389

## Citation

> US National Institutes of Health, RePORTER application 10876389, Characterization of the RRS: a new chromosomal structural element in E. coli (5R21AI179102-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10876389. Licensed CC0.

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