DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I.
SummaryTranscription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R-loops. In this paper, we have used several strategies to study the effects of elongation/ termination properties of RNA polymerase on such transcription-induced supercoiling. Effects on R-loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R-loop-dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R-loop-dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R-loop-dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R-loop-dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in Rloop formation in Escherichia coli . Finally, our results also indicate that what determines the steady-state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription-translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.
SummaryOne major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop. The goal of the present study was to identify other potential regulators of Rloop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identi®ed the topB gene encoding DNA topoisomerase III as an ef®cient suppressor of topA null mutations and, hence, of R-loop formation. We show that DNA topoisomerase III is able to relax transcriptioninduced negative supercoiling both in vitro and in vivo. An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loopdependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants ef®ciently. This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.
SummaryGyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought.
In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.
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