Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Apr 11;92(8):3526–3530. doi: 10.1073/pnas.92.8.3526

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.

M Drolet 1, P Phoenix 1, R Menzel 1, E Massé 1, L F Liu 1, R J Crouch 1
PMCID: PMC42200  PMID: 7536935

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 of genetic 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 of RNase 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.

Full text

PDF
3526

Images in this article

3528Fig. 1
on p.3528
3530Fig. 3
on p.3530

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bockrath R., Wolff L., Farr A., Crouch R. J. Amplified RNase H activity in Escherichia coli B/r increases sensitivity to ultraviolet radiation. Genetics. 1987 Jan;115(1):33–40. doi: 10.1093/genetics/115.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. DiNardo S., Voelkel K. A., Sternglanz R., Reynolds A. E., Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. doi: 10.1016/0092-8674(82)90403-2. [DOI] [PubMed] [Google Scholar]
  3. Drolet M., Wu H. Y., Liu L. F. Roles of DNA topoisomerases in transcription. Adv Pharmacol. 1994;29A:135–146. doi: 10.1016/s1054-3589(08)60543-8. [DOI] [PubMed] [Google Scholar]
  4. Dürrenberger M., Bjornsti M. A., Uetz T., Hobot J. A., Kellenberger E. Intracellular location of the histonelike protein HU in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4757–4768. doi: 10.1128/jb.170.10.4757-4768.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Itaya M., Crouch R. J. Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H. Mol Gen Genet. 1991 Jul;227(3):433–437. doi: 10.1007/BF00273934. [DOI] [PubMed] [Google Scholar]
  6. Jin D. J., Burgess R. R., Richardson J. P., Gross C. A. Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1453–1457. doi: 10.1073/pnas.89.4.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kanaya S., Crouch R. J. Low levels of RNase H activity in Escherichia coli FB2 rnh result from a single-base change in the structural gene of RNase H. J Bacteriol. 1983 May;154(2):1021–1026. doi: 10.1128/jb.154.2.1021-1026.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kirkegaard K., Wang J. C. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J Mol Biol. 1985 Oct 5;185(3):625–637. doi: 10.1016/0022-2836(85)90075-0. [DOI] [PubMed] [Google Scholar]
  9. Kogoma T. Escherichia coli RNA polymerase mutants that enhance or diminish the SOS response constitutively expressed in the absence of RNase HI activity. J Bacteriol. 1994 Mar;176(5):1521–1523. doi: 10.1128/jb.176.5.1521-1523.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kogoma T., Hong X., Cadwell G. W., Barnard K. G., Asai T. Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities. Biochimie. 1993;75(1-2):89–99. doi: 10.1016/0300-9084(93)90029-r. [DOI] [PubMed] [Google Scholar]
  11. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ogawa T., Pickett G. G., Kogoma T., Kornberg A. RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1040–1044. doi: 10.1073/pnas.81.4.1040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pruss G. J., Drlica K. Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8952–8956. doi: 10.1073/pnas.83.23.8952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pruss G. J., Franco R. J., Chevalier S. G., Manes S. H., Drlica K. Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants. J Bacteriol. 1986 Oct;168(1):276–282. doi: 10.1128/jb.168.1.276-282.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Raji A., Zabel D. J., Laufer C. S., Depew R. E. Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. J Bacteriol. 1985 Jun;162(3):1173–1179. doi: 10.1128/jb.162.3.1173-1179.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sternglanz R., DiNardo S., Voelkel K. A., Nishimura Y., Hirota Y., Becherer K., Zumstein L., Wang J. C. Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition. Proc Natl Acad Sci U S A. 1981 May;78(5):2747–2751. doi: 10.1073/pnas.78.5.2747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Subia N. L., Kogoma T. Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity. J Mol Biol. 1986 Jun 5;189(3):389–399. doi: 10.1016/0022-2836(86)90311-6. [DOI] [PubMed] [Google Scholar]
  18. Torrey T. A., Atlung T., Kogoma T. dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants. Mol Gen Genet. 1984;196(2):350–355. doi: 10.1007/BF00328070. [DOI] [PubMed] [Google Scholar]
  19. Trucksis M., Depew R. E. Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2164–2168. doi: 10.1073/pnas.78.4.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  21. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES