Pap1+ confers microtubule damage resistance to mut2a, an extragenic suppressor of the rad26:4A allele in S. pombe.

Document Type : Original article

Authors

1 Department of Biology, University of Colorado Colorado Springs, 1420 Austin Bluffs Parkway Colorado Springs, CO 80918

2 Pine Creek high school, 10750 Thunder Mountain Ave, Colorado Springs, CO 80908

Abstract

The DNA structure checkpoint protein Rad26ATRIP is also required for an interphase microtubule damage response. This checkpoint delays spindle pole body separation and entry into mitosis following treatment of cells with microtubule poisons. This checkpoint requires cytoplasmic Rad26ATRIP, which is compromised by the rad26:4A allele that inhibits cytoplasmic accumulation of Rad26ATRIP following microtubule damage. The rad26::4a allele also disrupts minichromosome stability and cellular morphology, suggesting that the interphase microtubule damage checkpoint pathway operates in an effort to maintain chromosome stability and proper cell shape. To identify other proteins of the Rad26-dependent interphase microtubule damage response, we used ultra violet (UV) radiation to identify extragenic interaction suppressors of the rad26::4A growth defect on microtubule poisons. One suppressor mutation, which we named mut2a, permitted growth of rad26:4A cells on MBC media and conferred sensitivity to a microtubulin poison upon genetic outcross. In an attempt to clone this interaction suppressor using a genomic library complementation strategy, we instead isolated pap1+as an extracopy suppressor of the mut2a growth defect. We discuss the mechanism by which pap1+ overexpression may allow growth of mut2a cells in conditions that destabilize microtubules. 

Keywords

References

1. Carr AM. DNA structure checkpoints in fission yeast. Semin Cell Biol 1995;6:65-72.
2. Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Mol Cell 2010;40:179-204.
3. Bentley NJ, Hawkins G, Keegan K, DeMaggio A, Holtzman D, Ford JC, Hoekstra M, Carr AM. The Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J 1996;15:6641-6651.
4. Edwards RJ, Bentley NJ, Carr AM. A Rad3–Rad26 complex responds to DNA damage independently of other checkpoint proteins. Nat Cell Biol 1999;1:393-398.
5. Wolkow TD, Enoch T. Fission yeast Rad26 responds to DNA damage independently of Rad3. BMC Genetics 2003 ;4:1471-2156.
6. Mcgowan CH, Russell P. The DNA damage response: Sensing and signaling. Current Opinion in Cell Biology 2004;16:629-633.
7. Zou L. Checkpoint Mec-tivation Comes in Many Flavors. Mol Cell 2009;36:734-735.
8. Saka Y, Esashi F, Matsusaka T, Mochida S, Yanagida M. Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. Genes Dev 1997;11:3387-3400.
9. Tanaka K, Russell P. Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nature Cell Biology 2001;3:966-972.
10. Xu Y. Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast. Genes & Development 2006;20:990-1003.
11. Yue M, Zeng L, Singh A, Xu Y. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe. PLoS One 2014;9.
12. Baschal EE, Chen KJ, Elliott LG, Herring MJ, Verde SC, Wolkow TD. The fission yeast DNA structure checkpoint protein RAD26ATRIP/LCDI/UVSD accumulates in the cytoplasm following microtubule destabilization. BMC Cell Biol 2006;7:32.
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. Yamamoto M. Genetic analysis of resistant mutants to antimitotic benzimidazole compounds in Schizosaccharomyces pombe. Mol Gen Genet 1980;180:231-234.
15. Umesono K, Toda T, Hayashi S, Yanagida M. Cell division cycle genes nda2 and nda3 of  the  fission  yeast  Schizosaccharomyces  pombe control  microtubular organization  and  sensitivity  to  anti-mitotic  benzimidazole  compounds.  J Mol Biol 1983;168:271-284.
16. Balestra FR, Jimenez J. A G2-phase microtubule-damage response in fission yeast. Genetics 2008;180:2073-2080.
17. Forsburg SL. The art and design of genetic screens: yeast. Nat Rev Genet 2001;2:659-668.
18. Martinho RG, Carr AM. Isolation of DNA structure-dependent checkpoint mutants in S. pombe. In: Henderson D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 113. Humana Press, 1999.
19. Bähler J, Wu J, Longtine MS, Shah NG, Iii AM, 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. Nakase Y, Nakamura T, Hirata A, Routt SM, Skinner HB, Bankaitis VA, Shimoda C. The Schizosaccharomyces pombe spo20 gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation. Mol Biol Cell 2001;12:901-917.
21. Moreno S, Klar A, Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods in Enzymol 1991;795-823.
22. Walfridsson J. The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres. Nucleic Acids Res 2005;33:2868-2879.
23. Penney M, Samejima I, Wilkinson CR, Mcinerny CJ, Mathiassen SG, Wallace M, Gordon C. Fission yeast 26S proteasome mutants are multi-drug resistant due to stabilization of the Pap1 transcription factor. PLoS One 2012;7.
24. Toda T, Shimanuki M, Yanagida M. Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev 1991;5: 60-73.
25. Losson R, Lacroute F. Plasmids carrying the yeast OMP decarboxylase structural and regulatory genes: Transcription regulation in a foreign environment. Cell 1983;32:371-377.
26. Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, Sekido S, Yoshida M. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nature Biotechnology 2006;24:841-7.
27. Matsuyama A, Shirai A, Yashiroda Y, Kamata A, Horinouchi S, Yoshida M.  PDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast. Yeast 2004;21:1289-1305.
28. Toone WM, Kuge S, Samuels M, Morgan BA, Toda T, Jones N. Regulation of the fission yeast transcription factor Pap1 by oxidative stress: Requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1. Genes Dev 1998;12:1453-1463.
29. Quinn J. Distinct Regulatory Proteins Control the Graded Transcriptional Response to Increasing H2O2 Levels in Fission Yeast Schizosaccharomyces pombe. Mol Biol Cell 2002; 13:805-816.
30. Belfield C, Queenan C, Rao H, Kitamura K, Walworth NC. The oxidative stress responsive transcription factor Pap1 confers DNA damage resistance on checkpoint-deficient fission yeast cells. PLoS One 2014;9.
31. Calvo IA, Gabrielli N, Iglesias-Baena I, García-Santamarina S, Hoe K, Kim DU, Hidalgo E. Genome-wide screen of genes required for caffeine tolerance in fission yeast. PLoS One 2009;4.

Files

Share

How to cite

Statistics