Monitoring the DNA Topoisomerase II Checkpoint in Saccharomyces cerevisiae

  • Katherine Furniss
  • Amit C. J. Vas
  • Andrew B. Lane
  • Duncan J. Clarke
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control which responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.

Key words

Topoisomerase II checkpoint Anaphase Spindle elongation Chromosome condensation Budding yeast 

References

  1. 1.
    Porter AC, Farr CJ (2004) Topoisomerase II: untangling its contribution at the centromere. Chromosome Res 12:569–583CrossRefPubMedGoogle Scholar
  2. 2.
    Warburton PE, Earnshaw WC (1997) Untangling the role of DNA topoisomerase II in mitotic chromosome structure and function. Bioessays 19:97–99CrossRefPubMedGoogle Scholar
  3. 3.
    Holm C, Stearns T, Botstein D (1989) DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol Cell Biol 9:159–168CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Uemura T, Ohkura H, Adachi Y, Morino K, Shiozaki K, Yanagida M (1987) DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. Pombe. Cell 50:917–925CrossRefPubMedGoogle Scholar
  5. 5.
    Adachi Y, Luke M, Laemmli UK (1991) Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell 64:137–148CrossRefPubMedGoogle Scholar
  6. 6.
    Wood ER, Earnshaw WC (1990) Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II. J Cell Biol 111:2839–2850CrossRefPubMedGoogle Scholar
  7. 7.
    Roberge M, Th'ng J, Hamaguchi J, Bradbury EM (1990) The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells. J Cell Biol 111:1753–1762CrossRefPubMedGoogle Scholar
  8. 8.
    Newport J, Spann T (1987) Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell 48:219–230CrossRefPubMedGoogle Scholar
  9. 9.
    Vas AC, Andrews CA, Kirkland Matesky K, Clarke DJ (2007) In vivo analysis of chromosome condensation in Saccharomyces Cerevisiae. Mol Biol Cell 18:557–568CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Clarke DJ, Vas AC, Andrews CA, Diaz-Martinez LA, Gimenez-Abian JF (2006) Topoisomerase II checkpoints: universal mechanisms that regulate mitosis. Cell Cycle 5:1925–1928CrossRefPubMedGoogle Scholar
  11. 11.
    Andrews CA, Vas AC, Meier B, Gimenez-Abian JF, Diaz-Martinez LA, Green J, Erickson SL, Vanderwaal KE, Hsu WS, Clarke DJ (2006) A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin. Genes Dev 20:1162–1174CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Skoufias DA, Lacroix FB, Andreassen PR, Wilson L, Margolis RL (2004) Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase. Mol Cell 15:977–990CrossRefPubMedGoogle Scholar
  13. 13.
    Mikhailov A, Shinohara M, Rieder CL (2004) Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J Cell Biol 166:517–526CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gimenez-Abian JF, Weingartner M, Binarova P, Clarke DJ, Anthony RG, Calderini O, Heberle-Bors E, de la Moreno Diaz ES, Bogre L, De la Torre C (2002) A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2. Cell Cycle 1:187–192CrossRefPubMedGoogle Scholar
  15. 15.
    Deming PB, Flores KG, Downes CS, Paules RS, Kaufmann WK (2002) ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase. J Biol Chem 277:36832–36838CrossRefPubMedGoogle Scholar
  16. 16.
    Deming PB, Cistulli CA, Zhao H, Graves PR, Piwnica-Worms H, Paules RS, Downes CS, Kaufmann WK (2001) The human decatenation checkpoint. Proc Natl Acad Sci U S A 98:12044–12049CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Downes CS, Clarke DJ, Mullinger AM, Giménez-Abián JF, Creighton AM, Johnson RT (1994) A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 372:467–470CrossRefPubMedGoogle Scholar
  18. 18.
    Goto T, Wang JC (1984) Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell 36:1073–1080CrossRefPubMedGoogle Scholar
  19. 19.
    Straight AF, Marshall WF, Sedat JW, Murray AW (1997) Mitosis in living budding yeast: anaphase a but no metaphase plate. Science 277:574–578CrossRefPubMedGoogle Scholar
  20. 20.
    Winey M, Mamay CL, O'Toole ET, Mastronarde DN, Giddings TH Jr, McDonald KL, McIntosh JR (1995) Three-dimensional ultrastructural analysis of the Saccharomyces Cerevisiae mitotic spindle. J Cell Biol 129:1601–1615CrossRefPubMedGoogle Scholar
  21. 21.
    Pearson CG, Maddox PS, Salmon ED, Bloom K (2001) Budding yeast chromosome structure and dynamics during mitosis. J Cell Biol 152:1255–1266CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Straight AF, Belmont AS, Robinett CC, Murray AW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608CrossRefPubMedGoogle Scholar
  23. 23.
    Richardson HE, Wittenberg C, Cross FR, Reed SI (1989) An essential G1 function for cyclin-like proteins in yeast. Cell 59:1127–1133CrossRefPubMedGoogle Scholar
  24. 24.
    Clarke DJ, Segal M, Mondesert G, Reed SI (1999) The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast. Curr Biol 9:365–368CrossRefPubMedGoogle Scholar
  25. 25.
    Furniss K, Tsai H-J, Byl JAW, Lane AB, Vas AC, Hsu WS, Osheroff N, Clarke DJ (2013) Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation. PLoS Genetics 9(10):e1003832CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Haase SB, Reed SI (2002) Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1:132–136CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Katherine Furniss
    • 1
    • 2
  • Amit C. J. Vas
    • 1
    • 3
  • Andrew B. Lane
    • 1
  • Duncan J. Clarke
    • 1
  1. 1.Department of Genetics, Cell Biology & DevelopmentUniversity of MinnesotaMinneapolisUSA
  2. 2.St. John’s UniversityCollegevilleUSA
  3. 3.Biotechnology R&DCargill, Inc.PlymouthUSA

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