Genomic Landscape of Single-stranded DNA Gapped Intermediates in Escherichia coli

Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during normal and aberrant DNA replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert to C to U on ssDNA, combined with deep sequencing, we have mapped genomic ssDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase before and after UV radiation, and in stationary phase cells. Notably, a monotonic reduction in ssDNA occurred symmetrically between the origin of DNA replication (OriC) and the replication terminus (Ter) for log phase cells, a hallmark feature of DNA replication. In contrast, stationary phase cells showed no indication of an OriC to Ter ssDNA gradient. More generally, we have identified a spatially diverse landscape containing thousands of highly enriched hot ssDNA regions along with smaller numbers of cold regions. This analysis can be used under 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.