Roles of Type 1A Topoisomerases in Genome Maintenance in <i>Escherichia coli</i>

<div><p>In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the <i>E. coli</i> topo I (<i>topA</i>) and III (<i>topB</i>) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that <i>topA</i> mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting <i>recA</i> or <i>recQ</i> but not by deleting <i>recJ</i> or <i>recO</i>, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-<i>recQ</i> deletion and topo IIII overproduction are epistatic to <i>recA</i> deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an <i>oriC</i>::<i>aph</i> suppressor mutation which is <i>oriC</i>-competent in <i>topA</i> null but not in isogenic <i>topA⁺</i> cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double <i>topA rnhA</i> mutants affecting constitutive stable DNA replication, <i>dnaT</i>::<i>aph</i> and <i>rne</i>::<i>aph</i>, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from <i>oriC</i> (topo I alone) and R-loops and (ii) by acting with RecQ.</p>