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

Abstract

It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.Escherichia coli DNA topoisomerase I, a member of the type IA family of topoisomerases, specifically relaxes negatively supercoiled DNA (1, 2). This specificity is explained by the fact that this enzyme binds to the junction of single-stranded and double-stranded DNA regions. DNA opening, and hence the generation of single-stranded DNA regions, is promoted by negative but not positive supercoiling. Hot spots for relaxation by DNA topoisomerase I are provided during transcription elongation in the frame of the twin-domain model (3). Indeed, very high levels of negative supercoiling can be generated behind the moving RNA polymerase during transcription elongation (4, 5). An R-loop, in which the template strand is paired with the nascent RNA, leaving the nontemplate strand unpaired, also provides a hot spot for relaxation by this enzyme (6).The accumulated evidence over the last few years has allowed us to conclude that a major function of DNA topoisomerase I in E. coli is to inhibit R-loop formation during transcription elongation. 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). The finding that several topA null mutants carry compensatory gyr mutations (in gyrA or gyrB) that reduce DNA gyrase activity and correct their growth defect (9, 10) was therefore explained by the supercoiling activity of DNA gyrase, which promotes R-loop formation (7). On the contrary, DNA topoisomerase I activity inhibits R-lo...

Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes

“…Primer sets were as follows: rDNA1 (nucleotide [nt] positions 3 to 213 relative to ϩ1 for the rRNA transcript), 5Ј (5Ј-GCGAAAGCAGTTGAAGACAAGTTCG-3Ј) and 3Ј (5Ј-CACACTCTGG GAATTTCTTCCC-3Ј); rDNA2 (positions 1061 to 1274), 5Ј (5Ј-CGGGGAAT AAGGGTTCGATTCCGG-3Ј) and 3Ј (5Ј-CGGCTGCTGGCACCAGACTTG CCC-3Ј); rDNA3 (positions 2051 to 2250), 5Ј (5Ј-GGTGCTAGCATTTGCTG GTTATCC-3Ј) and 3Ј (5Ј-GCTCTATCCCCAGCACGACGGAG-3Ј); rDNA4 (positions 3001 to 3214), 5Ј (5Ј-GCCTGTTTGAGCGTCATTTCCTTC-3Ј) and 3Ј (5Ј-CGATAACGTTCCAATACGCTCAG-3Ј); rDNA5 (positions 3867 to 4074), 5Ј (5Ј-GGCATAATGGTTATATGCCGCCCG-3Ј) and 3Ј (5Ј-GCATAG TTCACCATCTTTCGGGTC-3Ј); rDNA6 (positions 5007 to 5224), 5Ј (5Ј-CAG CACCTTTGCTGGCTCCGGTGC-3Ј) and 3Ј (5Ј-CTGACCAAGGCCCTCAC TACCCG-3Ј); rDNA7 (positions 5851 to 6045), 5Ј (5Ј-CAGGTGGGGAGTTT GGCTGGGGCG-3Ј) and 3Ј (5Ј-CCTCTAGCCTCAAATTCCGAGGG-3Ј); VOL. 31,2011 TOPOISOMERASES AND rDNA TRANSCRIPTION 483 rDNA8 (positions 6607 to 6825), 5Ј (5Ј-GTTACGATCTGCTGAGATTAAGC C-3Ј) and 3Ј (5Ј-GGTACACTCTTACACACTATCATCC-3Ј); NTS1 (positions Ϫ2189 to Ϫ1912), 5Ј (5Ј-GAAAGGATTTGCCCGGACAGTTTG-3Ј) and 3Ј (5Ј-CTTCTTCCCAGTAGCCTGTTCCTT-3Ј); NTS2 (positions Ϫ265 to Ϫ26), 5Ј (5Ј-GTATGTTTTGTATGTTCCCGCGCG-3Ј) and 3Ј (5Ј-CATGAAGTAC CTCCCAACTACTTTTCCTC-3Ј); and ACT1, 5Ј (5Ј-CCAATTGCTCGAGAG ATTTC-3Ј) and 3Ј (5Ј-CATGATACCTTGGTGTCTTG-3Ј).…”

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.…”

Replication Fork Stalling at Natural Impediments

“…It is therefore believed that the deleterious effects of R loops in the topA strain are responsible for its phenotype. RNase H overproduction was also shown to correct the main known defect of the topA strain, a reduced rate of transcription elongation (106). Therefore, it is believed that transcriptional blocks at R loops (when RNA polymerase encounters an R loop formed by a preceding RNA polymerase) result in the low rate of transcription and, ultimately, poor growth.…”