DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation
protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on
RNA-
DNA hybrids (R-loops), which are normally removed by
enzymes such as
RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in
RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on
genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The
DNA copy number profiles of various
RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of
RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for
genome stability, offering a new understanding of how
RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.