Topoisomerase III limits RecA-dependent DNA amplification in the chromosome terminus with RecG

Topoisomerase (topo) III is a ubiquitous type IA enzyme whose role in suppressing inappropriate recombination is well established in eukaryotes but remains poorly defined in prokaryotes. In Escherichia coli, deletion of topB (encoding topo III) in topA mutants (topo I, which normally prevents R-loop formation) leads to pronounced Ter/Tus barrier-dependent DNA amplification in the chromosomal terminus region. In recG mutants, this phenotype was recently attributed to RecA-mediated D-loop formation at DNA double-strand ends (DSEs) generated at Ter/Tus barriers, triggering replication restart in the incorrect orientation (re-replication). Here, our data indicate that, as in recG mutants, RecA drives repeated cycles of Ter/Tus barrier-dependent DSE formation and re-replication, thereby promoting chromosomal terminus DNA amplification in topA topB cells. Deletion of topB enhances Ter/Tus-dependent amplification in rnhA cells but not in recG cells. Moreover, deletion of recG or topB, but not recQ, markedly exacerbates the replication-completion defect of topA mutants, unless recA is deleted or topo IV is overproduced. Together, these findings suggest that topo III and RecG cooperate to restrain RecA-mediated re-replication at sites of DSE formation, thereby preventing pathological DNA amplification.