Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I.

Abstract

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces ofgenetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression ofRNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.DNA topoisomerase (TOPO) I, originally known as o, is the major DNA relaxing activity in Escherichia coli (1). The gene encoding E. coli DNA TOPO I topA was localized to the cys-trp region of the chromosome (2, 3). Deletions that remove both cysB and topA were identified in E. coli strains and it was first assumed that topA was not an essential gene (3). A more careful analysis showed that a deletion of the topA gene could only be inherited in strains that contained a second mutation that would compensate for the loss of topA (4, 5). These studies identified mutations in both gyrA and gyrB that could compensate for the loss of topA and demonstrated that these mutations result in reduced global DNA supercoiling. This lead to a picture in which a "global balance" of DNA supercoiling is essential and controlled by the competing activities of DNA gyrase and TOPO I.In vitro DNA TOPO I does not relax negatively supercoiled DNA to completion (1). Other in vitro studies show that the specificity for negatively supercoiled DNA derives from a requirement of a short single-stranded DNA region as part of the enzyme-DNA complex (6). Since negative DNA supercoiling favors the unpairing of DNA strands, DNA molecules with high negative superhelical density will be a better substrate for DNA TOPO I. Some in vivo studies also suggest that DNA TOPO I does not relax DNA efficiently: in one study a topA mutation had little effect on the rate of in vivo DNA relaxation after the inhibition of DNA gyrase by coumermycin (7); and in a second in vivo study, DNA TOPO I produced only slow and partial DNA relaxation (8). However, a defect in DNA TOPO I can cause an in vivo increase in the level of negative supercoiling (5). Thus, these results suggest that DNA TOPO I functions to prevent DNA supercoiling from reaching an unacceptably high level.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3526Hypernegatively supercoiled DNA can, under certain circumstances, be produced during transcription (9, 10). In...

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

“…DSBs seem to be particularly frequent in the rDNA locus. Thus, R-loops and DSBs could stimulate recombination-driven replication events as observed in Candiada albicans mtDNA (42) growth and rDNA transcription (51)(52)(53), and that RNase H1 overexpression can partially compensate for the absence of Top1 (54). Other factors that possibly contribute to synthetic lethality include the accumulation of positive supercoiling generated ahead of RNAPI and in front of an advancing replication fork in convergent orientation.…”

RNase H Overproduction Corrects a Defect at the Level of Transcription Elongation during rRNA Synthesis in the Absence of DNA Topoisomerase I in Escherichia coli

“…The fact that topA null mutants are sensitive to changes in environmental conditions (7, 14 -17) may suggest that gene expression is somehow impaired by R-loop formation. For example, the finding that RNase H overproduction allows topA null mutants to more rapidly adapt to fresh media and to nutritional shift-ups, may suggest that R-loops inhibit the expression of genes required for such growth transitions (7,17). Interestingly, we have shown previously that R-loop formation can occur during transcription of a DNA fragment carrying a portion of the rrnB operon on a plasmid DNA, in the absence of DNA topoisomerase I (12).…”

“…Indeed, the growth problem of topA (encoding DNA topoisomerase I) null mutants was shown to be partially corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop (7). A correlation was also established between the level of DNA gyrase activity, the enzyme that introduces negative supercoiling within the chromosomal DNA, and the amount of RNase H required to stimulate the growth of topA null mutants (7) and to inhibit R-loop formation during transcription (8).…”

Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIβ

“…In addition, R-loop formation has been associated with recombination events that occur at mammalian immunoglobulin class switch sequences (27,28). Interestingly, it has been proposed that one of the primary roles of Escherichia coli topo I is to suppress R-loop formation during transcriptional elongation that could otherwise impair cell growth (29,30). In the absence of topo I, such as a topA mutant, extensive R-loop formation occurs, especially when the cells are grown at low temperature (31).…”