Abstract
Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.
Highlights
DNA topoisomerases are nicking-closing enzymes that solve the topological problems inherent to the double-helical structure of the DNA [1]
TopA topB null transductants in strains carrying a gyrase compensatory mutation could be obtained after 48 hours of incubation [31, 32]. These results demonstrated that E. coli cells can survive without type 1A topos but did not show if compensatory mutations were required for viability, and how such mutants could survive
Our results reveal three important findings: 1- Type 1A topos from both subfamilies can inhibit R-loop formation/accumulation mostly to prevent unregulated replication from R-loops. 2- High levels of replication from R-loops in the terminus region of the circular chromosome appear to be largely responsible for the strong phenotypes of topA topB null mutants. 3- topA topB null mutants are able to grow owing to the amplification of a chromosomal DNA region carrying the parC and parE genes that leads to topo IV overproduction
Summary
DNA topoisomerases (topos) are nicking-closing enzymes that solve the topological problems inherent to the double-helical structure of the DNA [1]. Type 1A topos are the sole ubiquitous topos, being present in the three domains of life [2, 3] They require single-stranded DNA (ssDNA) for binding, a substrate that is mostly provided by negatively supercoiled DNA for some of these enzymes, as is the case for bacterial topos I, the members of the first type 1A topo subfamily, encoded by topA [1]. Topo IV, at its wild-type cellular level, does play a role in the regulation of supercoiling in E. coli [9] These observations lead to a model in which the chromosomal negative supercoiling in the cell is set by the opposing enzymatic activities of DNA gyrase, introducing negative supercoiling, and topo I and IV relaxing it [9]. Topo I physically interacts with RNAP in E. coli [11, 12] and this interaction is found between M. tuberculosis topo I and RNAP, though in that case by a distinct mechanism [13]
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