Fission yeast Ase1PRC1 is required for the G2-microtubule damage response

Document Type : Original article


Department of Biology, University of Colorado at Colorado Springs, Colorado Springs, CO 80918


Schizosaccharomyces pombe delays entry into mitosis following G2 microtubule damage. This pathway is dependent on Rad26ATRIP, the regulatory subunit of the Rad26ATRIP/Rad3ATR DNA damage response (DDR) complex. However, this G2 microtubule damage response pathway acts independently of the G2 DNA damage checkpoint pathway. To identify other proteins in this G2 microtubule damage pathway, we previously screened a cDNA overexpression library for genes that rescued the sensitivity of rad26Δ cells to the microtubule poison thiabendazole. A partial cDNA fragment encoding only the C-terminal regulatory region of the microtubule bundling protein Ase1PRC1 was isolated. This fragment lacks the Ase1PRC1 dimerization and microtubule binding domains and retains the conserved C-terminal unstructured regulatory region. Here, we  report that ase1Δ cells fail to delay entry into mitosis following G2 microtubule damage. Microscopy revealed that Rad26ATRIP foci localized alongside Ase1PRC1 filaments, although we suggest that this is related to microtubule-dependent double strand break mobility that facilitates homologous recombination events. Indeed, we report that the DNA repair protein Rad52 co-localizes with Rad26ATRIP at these foci, and that localization of Rad26ATRIP to these foci depends on a Rad26ATRIP N-terminal region containing a checkpoint recruitment domain. To our knowledge, this is the first report implicating Ase1PRC1 in regulation of the G2/M transition.


  1. Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 2010;79:181-211.
  2. Jasin M, Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 2013;5:a012740.
  3. Shiloh Y. The ATM-mediated DNA-damage response: taking shape. Trends Biochem Sci 2006;31:402-410.
  4. Maréchal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb Perspect Biol 2013;5:a012716.
  5. Pannunzio NR, Watanabe G, Lieber MR. Nonhomologous DNA end-joining for repair of DNA double-strand breaks. J Biol Chem 2018;293:10512-10523.
  6. Burger K, Ketley RF, Gullerova M. Beyond the Trinity of ATM, ATR, and DNA-PK: multiple kinases shape the DNA damage response in concert with RNA metabolism. Front Mol Biosci 2019;6:61.
  7. Trielli MO, Andreassen PR, Lacroix FB, Margolis RL. Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific-related mortality of transformed cells. J Cell Biol 1996;135:689-700.
  8. Giannakakou P, Robey R, Fojo T, Blagosklonny MV. Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001;20:3806-3813.
  9. Mantel CR, Braun SE, Lee Y, Kim YJ, Broxmeyer HE. The interphase microtubule damage checkpoint defines an S-phase commitment point and does not require p21(waf-1). Blood 2001;97:1505-1507.
  10. Mantel CR, Gelfano VM, Kim YJ, McDaniel A, Lee Y, Boswell Hs, Broxmeyer HE. P21waf-1-Chk1 pathway monitors G1 phase microtubule integrity and is crucial for restriction transition. Cell Cycle 2002;1:327-336.
  11. Naaz F, Ahmad F, Lone BA, Pokharel YR, Fuloria NK, Fuloria S, Ravichandran M, Pattabhiraman L, Shafi S, Shahar Yar M. Design and synthesis of newer 1,3,4-oxadiazole and 1,2,4-triazole based Topsentin analogues as anti-proliferative agent targeting tubulin. Bioorg Chem 2020;95:103519.
  12. Cussiol JRR, Soares BL, Oliveira FMB de. From yeast to humans: Understanding the biology of DNA damage response (DDR) kinases. Genet Mol Biol 2019;43:e20190071.
  13. Herring M, Davenport N, Stephan K, Campbell S, White R, Kark J, Wolkow TD. Fission yeast Rad26ATRIP delays spindle-pole-body separation following interphase microtubule damage. J Cell Sci 2010;123:1537-1545.
  14. Balestra FR, Jimenez J. A G2-phase microtubule-damage response in fission yeast. Genetics 2008;180:2073-2080.
  15. Nurse P. Genetic control of cell size at cell division in yeast. Nature 1975;256:547-551.
  16. Russell P, Nurse P. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell 1987;49:559-567.
  17. Raleigh JM, O’Connell MJ. The G(2) DNA damage checkpoint targets both Wee1 and Cdc25. J Cell Sci 2000;113:1727–1736.
  18. Craven RA, Griffiths DJ, Sheldrick KS, Randall RE, Hagan IM, Carr AM. Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 1998; 221:59-68.
  19. Bähler J, Wu JQ, Longtine MS, Shah NG, McKenzie A, Steever AB, Wach A, Philippsen P, Pringle JR. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 1998;14:943-951.
  20. Bridge AJ, Morphew M, Bartlett R, Hagan IM. The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes Dev 1998;12:927-942.
  21. Baschal EE, Chen KJ, Elliott LG, Herring MJ, Verde SC, Wolkow TD. The fission yeast DNA structure checkpoint protein Rad26ATRIP/LCD1/UVSD accumulates in the cytoplasm following microtubule destabilization. BMC Cell Biol 2006;7:32.
  22. Yamashita A, Sato M, Fujita A, Yamamoto M, Toda T. The roles of fission yeast Ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling. Mol Biol Cell 2005;16:1378-1395.
  23. Schuyler SC, Liu JY, Pellman D. The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins. J Cell Biol 2003;160:517-528.
  24. Glotzer M. The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat Rev Mol Cell Biol 2009;10:9-20.
  25. Subramanian R, Wilson-Kubalek EM, Arthur CP, Bick MJ, Campbell EA, Darst SA, Milligan RA, Kapoor TM. Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. Cell 2010;142:433-443.
  26. She ZY, Wei YL, Lin Y, Li YL, Lu MH. Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly. Biol Rev Camb Philos Soc 2019;94:2033-2048.
  27. Fantes P. Epistatic gene interactions in the control of division in fission yeast. Nature 1979; 279:428-430.
  28. Hagan IM, Grallert A, Simanis V. Analysis of the Schizosaccharomyces pombe Cell Cycle. Cold Spring Harb Protoc 2016;2016.
  29. Akera T, Sato M, Yamamoto M. Interpolar microtubules are dispensable in fission yeast meiosis II. Nat Commun 2012;3:695.
  30. Hartmuth S, Petersen J. Fission yeast Tor1 functions as part of TORC1 to control mitotic entry through the stress MAPK pathway following nutrient stress. J Cell Sci 2009; 122:1737-1746.
  31. Loïodice I, Staub J, Setty TG, Nguyen N-PT, Paoletti A, Tran PT. Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast. Mol Biol Cell 2005;16:1756-1768.
  32. Wolkow TD, Enoch T. Fission yeast Rad26 responds to DNA damage independently of Rad3. BMC Genet 2003;4:6.
  33. Symington LS. Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol 2016;51:195-212.
  34. Du LL, Nakamura TM, Moser BA, Russell P. Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes. Mol Cell Biol 2003;23:6150-6158.
  35. Meister P, Poidevin M, Francesconi S, Tratner I, Zarzov P, Baldacci G. Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast. Nucleic Acids Res 2003;31:5064-5073.
  36. Swartz RK, Rodriguez EC, King MC. A role for nuclear envelope-bridging complexes in homology-directed repair. Mol Biol Cell 2014;25:2461-2471.
  37. De Souza CP, Ye XS, Osmani SA. Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans. Mol Biol Cell 1999;10:3661-3674.
  38. Namiki Y, Zou L. ATRIP associates with replication protein A-coated ssDNA through multiple interactions. Proc Natl Acad Sci U S A 2006;103:580-585.
  39. Ball HL, Ehrhardt MR, Mordes DA, Glick GG, Chazin WJ, Cortez D. Function of a conserved checkpoint recruitment domain in ATRIP proteins. Mol Cell Biol 2007; 27:3367-3377.
  40. Xu X, Vaithiyalingam S, Glick GG, Mordes DA, Chazin WJ, Cortez D. The basic cleft of RPA70N binds multiple checkpoint proteins, including RAD9, to regulate ATR signaling. Mol Cell Biol 2008;28:7345-7353.
  41. Rincon SA, Lamson A, Blackwell R, Syrovatkina V, Fraisier V, Paoletti A, Betterton MD, Tran PT. Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast. Nat Commun 2017;8:15286.
  42. Yukawa M, Kawakami T, Okazaki M, Kume K, Tang NH, Toda T. A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast. Mol Biol Cell 2017;28:3647-3659.
  43. Ebina H, Ji L, Sato M. CLASP promotes microtubule bundling in metaphase spindle independently of Ase1/PRC1 in fission yeast. Biol Open 2019;8:bio045716.
  44. Mollinari C, Kleman J-P, Jiang W, Schoehn G, Hunter T, Margolis RL. PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone. J Cell Biol 2002; 157:1175-1186.
  45. Vernì F, Somma MP, Gunsalus KC, Bonaccorsi S, Belloni G, Goldberg ML, Gatti M. Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis. Curr Biol 2004;14:1569-1575.
  46. Meadows JC, Millar J. Latrunculin a delays anaphase onset in fission yeast by disrupting an Ase1-independent pathway controlling mitotic spindle stability. Mol Biol Cell 2008; 19:3713-3723.
  47. Expósito-Serrano M, Sánchez-Molina A, Gallardo P, Salas-Pino S, Daga RR. Selective nuclear pore complex removal drives nuclear envelope division in fission yeast. Curr Biol 2020; 30:3212-3222.
  48. Fu C, Ward JJ, Loiodice I, Velve-Casquillas G, Nedelec FJ, Tran PT. Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation. Dev Cell 2009;17:257-267.
  49. Le Goff X, Woollard A, Simanis V. Analysis of the cps1 gene provides evidence for a septation checkpoint in Schizosaccharomyces pombe. Mol Gen Genet 1999;262:163-172.
  50. Liu J, Wang H, Balasubramanian MK. A checkpoint that monitors cytokinesis in Schizosaccharomyces pombe. J Cell Sci 2000;113:1223-1230.
  51. Mishra M, Karagiannis J, Trautmann S, Wang H, McCollum D, Balasubramanian MK. The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe. J Cell Sci 2004; 117:3897-3910.
  52. Trautmann S, McCollum D. Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation. Curr Biol 2005;15:1384-1389.