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. 2003 Jul;164(3):935–945. doi: 10.1093/genetics/164.3.935

Identification of a topoisomerase I mutant, scsA1, as an extragenic suppressor of a mutation in scaA(NBS1), the apparent homolog of human nibrin in Aspergillus nidulans.

Marcia R Z Kress Fagundes 1, Larissa Fernandes 1, Marcela Savoldi 1, Steven D Harris 1, Maria H S Goldman 1, Gustavo H Goldman 1
PMCID: PMC1462625  PMID: 12871905

Abstract

The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. Mutations in scaA(NBS1), which encodes the apparent homolog of human nibrin in Aspergillus nidulans, inhibit growth in the presence of the antitopoisomerase I drug camptothecin. This article describes the selection and characterization of extragenic suppressors of the scaA1 mutation, with the aim of identifying other proteins that interfere with the pathway or complex in which the ScaA would normally be involved. Fifteen extragenic suppressors of the scaA1 mutation were isolated. The topoisomerase I gene can complement one of these suppressors. Synergistic interaction between the scaA(NBS1) and scsA(TOP1) genes in the presence of DNA-damaging agents was observed. Overexpression of topoisomerase I in the scaA1 mutant causes increased sensitivity to DNA-damaging agents. The scsA(TOP1) and the scaA(NBS1) gene products could functionally interact in pathways that either monitor or repair DNA double-strand breaks.

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Selected References

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  1. Andoh T., Ishii K., Suzuki Y., Ikegami Y., Kusunoki Y., Takemoto Y., Okada K. Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5565–5569. doi: 10.1073/pnas.84.16.5565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballance D. J., Buxton F. P., Turner G. Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun. 1983 Apr 15;112(1):284–289. doi: 10.1016/0006-291x(83)91828-4. [DOI] [PubMed] [Google Scholar]
  3. Benedetti P., Fiorani P., Capuani L., Wang J. C. Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine. Cancer Res. 1993 Sep 15;53(18):4343–4348. [PubMed] [Google Scholar]
  4. Bennett R. J., Dunderdale H. J., West S. C. Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell. 1993 Sep 24;74(6):1021–1031. doi: 10.1016/0092-8674(93)90724-5. [DOI] [PubMed] [Google Scholar]
  5. Bergen L. G., Upshall A., Morris N. R. S-phase, G2, and nuclear division mutants of Aspergillus nidulans. J Bacteriol. 1984 Jul;159(1):114–119. doi: 10.1128/jb.159.1.114-119.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berger J. M. Structure of DNA topoisomerases. Biochim Biophys Acta. 1998 Oct 1;1400(1-3):3–18. doi: 10.1016/s0167-4781(98)00124-9. [DOI] [PubMed] [Google Scholar]
  7. Boddy M. N., Gaillard P. H., McDonald W. H., Shanahan P., Yates J. R., 3rd, Russell P. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell. 2001 Nov 16;107(4):537–548. doi: 10.1016/s0092-8674(01)00536-0. [DOI] [PubMed] [Google Scholar]
  8. Bruschi G. C., de Souza C. C., Fagundes M. R., Dani M. A., Goldman M. H., Morris N. R., Liu L., Goldman G. H. Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA+, related to the cellular DNA damage response. Mol Genet Genomics. 2001 Apr;265(2):264–275. doi: 10.1007/s004380000411. [DOI] [PubMed] [Google Scholar]
  9. Carney J. P., Maser R. S., Olivares H., Davis E. M., Le Beau M., Yates J. R., 3rd, Hays L., Morgan W. F., Petrini J. H. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell. 1998 May 1;93(3):477–486. doi: 10.1016/s0092-8674(00)81175-7. [DOI] [PubMed] [Google Scholar]
  10. Frei C., Gasser S. M. RecQ-like helicases: the DNA replication checkpoint connection. J Cell Sci. 2000 Aug;113(Pt 15):2641–2646. doi: 10.1242/jcs.113.15.2641. [DOI] [PubMed] [Google Scholar]
  11. Froelich-Ammon S. J., Osheroff N. Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J Biol Chem. 1995 Sep 15;270(37):21429–21432. doi: 10.1074/jbc.270.37.21429. [DOI] [PubMed] [Google Scholar]
  12. Gupta M., Fujimori A., Pommier Y. Eukaryotic DNA topoisomerases I. Biochim Biophys Acta. 1995 May 17;1262(1):1–14. doi: 10.1016/0167-4781(95)00029-g. [DOI] [PubMed] [Google Scholar]
  13. Hofmann A. F., Harris S. D. The Aspergillus nidulans musN gene encodes a RecQ helicase that interacts with the PI-3K-related kinase UVSB. Genetics. 2001 Dec;159(4):1595–1604. doi: 10.1093/genetics/159.4.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kisselev L. L., Buckingham R. H. Translational termination comes of age. Trends Biochem Sci. 2000 Nov;25(11):561–566. doi: 10.1016/s0968-0004(00)01669-8. [DOI] [PubMed] [Google Scholar]
  15. Knab A. M., Fertala J., Bjornsti M. A. A camptothecin-resistant DNA topoisomerase I mutant exhibits altered sensitivities to other DNA topoisomerase poisons. J Biol Chem. 1995 Mar 17;270(11):6141–6148. doi: 10.1074/jbc.270.11.6141. [DOI] [PubMed] [Google Scholar]
  16. Knab A. M., Fertala J., Bjornsti M. A. Mechanisms of camptothecin resistance in yeast DNA topoisomerase I mutants. J Biol Chem. 1993 Oct 25;268(30):22322–22330. [PubMed] [Google Scholar]
  17. Larsen A. K., Skladanowski A. Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death. Biochim Biophys Acta. 1998 Oct 1;1400(1-3):257–274. doi: 10.1016/s0167-4781(98)00140-7. [DOI] [PubMed] [Google Scholar]
  18. Levin N. A., Bjornsti M. A., Fink G. R. A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest. Genetics. 1993 Apr;133(4):799–814. doi: 10.1093/genetics/133.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Megonigal M. D., Fertala J., Bjornsti M. A. Alterations in the catalytic activity of yeast DNA topoisomerase I result in cell cycle arrest and cell death. J Biol Chem. 1997 May 9;272(19):12801–12808. doi: 10.1074/jbc.272.19.12801. [DOI] [PubMed] [Google Scholar]
  20. Nitiss J. L., Nitiss K. C., Rose A., Waltman J. L. Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage. J Biol Chem. 2001 May 15;276(28):26708–26714. doi: 10.1074/jbc.M102674200. [DOI] [PubMed] [Google Scholar]
  21. Nitiss J., Wang J. C. DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7501–7505. doi: 10.1073/pnas.85.20.7501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Osmani S. A., May G. S., Morris N. R. Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans. J Cell Biol. 1987 Jun;104(6):1495–1504. doi: 10.1083/jcb.104.6.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. PONTECORVO G., ROPER J. A., HEMMONS L. M., MACDONALD K. D., BUFTON A. W. J. The genetics of Aspergillus nidulans. Adv Genet. 1953;5:141–238. doi: 10.1016/s0065-2660(08)60408-3. [DOI] [PubMed] [Google Scholar]
  25. Paull T. T., Gellert M. Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev. 1999 May 15;13(10):1276–1288. doi: 10.1101/gad.13.10.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Petrini J. H. The mammalian Mre11-Rad50-nbs1 protein complex: integration of functions in the cellular DNA-damage response. Am J Hum Genet. 1999 May;64(5):1264–1269. doi: 10.1086/302391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pâques F., Haber J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999 Jun;63(2):349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reid R. J., Benedetti P., Bjornsti M. A. Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs. Biochim Biophys Acta. 1998 Oct 1;1400(1-3):289–300. doi: 10.1016/s0167-4781(98)00142-0. [DOI] [PubMed] [Google Scholar]
  29. Rubin E., Pantazis P., Bharti A., Toppmeyer D., Giovanella B., Kufe D. Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin. J Biol Chem. 1994 Jan 28;269(4):2433–2439. [PubMed] [Google Scholar]
  30. Samadashwily G. M., Raca G., Mirkin S. M. Trinucleotide repeats affect DNA replication in vivo. Nat Genet. 1997 Nov;17(3):298–304. doi: 10.1038/ng1197-298. [DOI] [PubMed] [Google Scholar]
  31. Semighini Camile P., von Zeska Kress Fagundes Márcia Regina, Ferreira Joseane Cristina, Pascon Renata Castiglioni, de Souza Goldman Maria Helena, Goldman Gustavo Henrique. Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans. Mol Microbiol. 2003 Jun;48(6):1693–1709. doi: 10.1046/j.1365-2958.2003.03539.x. [DOI] [PubMed] [Google Scholar]
  32. Semighini Camile Pizeta, Marins Mozart, Goldman Maria Helena S., Goldman Gustavo Henrique. Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl Environ Microbiol. 2002 Mar;68(3):1351–1357. doi: 10.1128/AEM.68.3.1351-1357.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shiloh Y. Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Annu Rev Genet. 1997;31:635–662. doi: 10.1146/annurev.genet.31.1.635. [DOI] [PubMed] [Google Scholar]
  34. Trujillo K. M., Yuan S. S., Lee E. Y., Sung P. Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95. J Biol Chem. 1998 Aug 21;273(34):21447–21450. doi: 10.1074/jbc.273.34.21447. [DOI] [PubMed] [Google Scholar]
  35. Van Dross R. T., Rao K. V., Eisenberg E., Sanders M. M. Cloning and characterization of the Aspergillus nidulans DNA topoisomerase I gene. Gene. 1997 Dec 12;203(2):169–174. doi: 10.1016/s0378-1119(97)00508-8. [DOI] [PubMed] [Google Scholar]
  36. Wu L., Hickson I. D. RecQ helicases and topoisomerases: components of a conserved complex for the regulation of genetic recombination. Cell Mol Life Sci. 2001 Jun;58(7):894–901. doi: 10.1007/PL00000909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van Heemst D., Swart K., Holub E. F., van Dijk R., Offenberg H. H., Goosen T., van den Broek H. W., Heyting C. Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51. Mol Gen Genet. 1997 May;254(6):654–664. doi: 10.1007/s004380050463. [DOI] [PubMed] [Google Scholar]

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