Since we used an experimental system that was
independent of ΔtopA compensatory mutations there might be a number of reasons for the observed differences. The available topB overexpression data suggest that ΔtopA cells suffer from strong topological defects. It is possible the gyrB203(ts) compensatory mutation alleviated some of these defects even at low temperature, which might enable increased levels of RNase HI to suppress the phenotype even further . Alternatively, the level of RNA:DNA hybrids might be very high. Since we did not Selleckchem Belinostat measure the expression level of our ΔproB::rnhA + directly, we cannot exclude the possibility that the rnhA expression level is not high enough for suppression of the ΔtopA phenotype. To investigate whether an RNA:DNA hybrid processing activity is important in the absence of Topo I we generated a ΔrnhA ΔtopA double mutant, as it was described before that topA rnhA double mutants are inviable even if topA is suppressed by strong suppressor mutations such as gyrB203(ts) . We noticed that ΔrnhA ΔtopA double mutants were not able to form white colonies on minimal medium,
which suggests that the deletion of rnhA indeed exacerbates the topA phenotype (Figure Semaxanib concentration 4A panel ii). We transformed the ptopA/ΔtopA ΔrnhA strain with our P araBAD topB overexpression plasmid to verify that the ΔrnhA ΔtopA double mutant can be partially suppressed by overexpression of topB, as reported . However, overexpression of topB did not suppress the synthetic lethality of ΔrnhA ΔtopA cells in our system (Figure 4B). Cells cannot grow in the absence of the topA plasmid despite the overexpression of topB. However, in cells retaining the topA plasmid the Prostatic acid phosphatase high levels of topoisomerase III is toxic, which explains the almost total absence of colonies (Figure 4B). Thus, the resolution of topological stress does
not render ΔtopA viable if the major enzyme that processes DNA:RNA hybrids is absent. Figure 4 rnhA and recG deletions exacerbate the ΔtopA phenotype. (A) No white colonies are observed on minimal medium when ΔtopA is combined with NVP-BEZ235 nmr either rnhA or recG. (B-C) Overexpression of topB in a ptopA + /topA recG background allows formation of white colonies. The overexpression of topB in ptopA + /topA rnhA cells has no effect Our results show that RecG can not compensate for the absence of RNase HI (Figure 2). However, if RecG processes some R-loops in vivo, the deletion of recG in a ΔtopA background should exacerbate the topA phenotype, as observed with rnhA. This was indeed observed. ΔrecG ΔtopA double mutants were not able to form any white colonies, neither on LB broth (data not shown) nor on minimal medium (Figure 4A panel iii).