Chromosomal replication is a source of spontaneous DNA double-strand breaks (DSBs). In E. coli, DSBs are repaired by homologous recombination using an undamaged sister template. During repair, the RecA protein polymerizes on single-stranded DNA generated at the site of the DSB and catalyses the search for sequence homologies on the undamaged sister template. This study utilized fluorescence microscopy to investigate the spatial and temporal dynamics of the RecA protein at the site of a replication-dependent DSB generated at the lacZ locus of the E. coli chromosome. The DSB was generated by SbcCD-mediated cleavage of a hairpin DNA structure formed on the lagging strand template of the replication fork by a long palindromic sequence. The tandem insertion of a recA-mCherry gene with the endogenous recA gene at the natural chromosomal locus produced no detectable effect on cell viability in the presence of DSB formation. During repair, the fluorescently-labelled RecA protein formed a transient focus, which was inferred to be the RecA nucleoprotein filament at the site of the replication-dependent DSB. The duration of the RecA focus at the site of the DSB was modestly reduced in a ΔdinI mutant and modestly increased in a ΔuvrD or ΔrecX mutant. Most cells underwent a period of extended cohesion of the sister lacZ loci after disappearance of the RecA focus. Segregation of the sister lacZ loci was followed by cell division, with each daughter cell obtaining a copy of the fluorescently-labelled lacZ locus. The RecA focus at the site of the DSB was observed predominantly between the mid-cell and the 1⁄4 position. In the absence of DSB formation, the lacZ locus exhibited dynamic movement between the mid-cell and the 1⁄4 position until the onset of segregation. Formation of the DSB and initiation of repair occurred at the spatial localization for replication of the lacZ locus while the downstream repair events occurred very close to the mid-cell. Genomic analysis of RecA-DNA interactions by ChIP-seq was used to demonstrate that the RecA focus at the lacZ locus was generated by the repair of the palindrome-induced DSB and not the repair of one-ended DSBs emanating from stalled replication forks at the repressor-bound operator arrays. This study has shown that the repair of a replication-dependent DSB occurs exclusively during the period of cohesion of the sister loci and the repair is efficiently completed prior to segregation of the two sister loci.