Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli

Abstract

Abstract Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert C to U on ssDNA, combined with deep sequencing, we have mapped gDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase in the presence and absence of UV irradiation, and in stationary phase cells. The fraction of ssDNA on gDNA is similar for W- and C-strands, ∼1.3% for log phase cells, ∼4.8% for irradiated log phase cells, and ∼8.5% for stationary phase cells. After UV irradiation, gaps increased in numbers and average lengths. A monotonic reduction in ssDNA occurred symmetrically between the DNA replication origin of (OriC) and terminus (Ter) for log phase cells with and without UV, a hallmark feature of DNA replication. Stationary phase cells showed no OriC → Ter ssDNA gradient. We have identified a spatially diverse gapped DNA landscape containing thousands of highly enriched ‘hot’ ssDNA regions along with smaller numbers of ‘cold’ regions. This analysis can be used for a wide variety of conditions to map ssDNA gaps generated when DNA metabolic pathways have been altered, and to identify proteins bound in the gaps.