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Genome Instability pp 375–385Cite as

Assays to Study Repair of Inducible DNA Double-Strand Breaks at Telomeres

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

The ends of linear chromosomes are constituted of repetitive DNA sequences called telomeres. Telomeres, nearby regions called subtelomeres, and their associated factors prevent chromosome erosion over cycles of DNA replication and prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). This raises the question of how cells repair DSBs that actually occur near chromosome ends. One approach is to edit the genome and engineer cells harboring inducible DSB sites within the subtelomeric region of different chromosome ends. This provides a reductionist and tractable genetic model system in which mechanisms mediating repair can be dissected via genetics, molecular biology, and microscopy tools.

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References

  1. Pfeiffer V, Lingner J (2013) Replication of telomeres and the regulation of telomerase. Cold Spring Harb Perspect Biol 5(5):a010405. doi:cshperspect.a010405 [pii]. doi:10.1101/cshperspect.a010405

    Article  PubMed  PubMed Central  Google Scholar 

  2. Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43(2 Pt 1):405–413. doi:0092-8674(85)90170-9 [pii]

    Article  CAS  PubMed  Google Scholar 

  3. Shampay J, Szostak JW, Blackburn EH (1984) DNA sequences of telomeres maintained in yeast. Nature 310(5973):154–157

    Article  CAS  PubMed  Google Scholar 

  4. Chan SW, Blackburn EH (2003) Telomerase and ATM/Tel1p protect telomeres from nonhomologous end joining. Mol Cell 11(5):1379–1387

    Article  CAS  PubMed  Google Scholar 

  5. Gottschling DE, Aparicio OM, Billington BL, Zakian VA (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63(4):751–762. doi:0092-8674(90)90141-Z [pii]

    Article  CAS  PubMed  Google Scholar 

  6. Mekhail K, Moazed D (2010) The nuclear envelope in genome organization, expression and stability. Nat Rev Mol Cell Biol 11(5):317–328. doi:nrm2894 [pii]. doi:10.1038/nrm2894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Moazed D (2001) Common themes in mechanisms of gene silencing. Mol Cell 8(3):489–498

    Article  CAS  PubMed  Google Scholar 

  8. Taddei A, Gasser SM (2012) Structure and function in the budding yeast nucleus. Genetics 192(1):107–129. doi:192/1/107 [pii]. doi:10.1534/genetics.112.140608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chan JN, Poon BP, Salvi J, Olsen JB, Emili A, Mekhail K (2011) Perinuclear cohibin complexes maintain replicative life span via roles at distinct silent chromatin domains. Dev Cell 20(6):867–879. doi:S1534-5807(11)00206-1 [pii]. doi:10.1016/j.devcel.2011.05.014

    Article  CAS  PubMed  Google Scholar 

  10. Taddei A, Hediger F, Neumann FR, Bauer C, Gasser SM (2004) Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. EMBO J 23(6):1301–1312. doi:10.1038/sj.emboj.7600144. 7600144 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bupp JM, Martin AE, Stensrud ES, Jaspersen SL (2007) Telomere anchoring at the nuclear periphery requires the budding yeast Sad1-UNC-84 domain protein Mps3. J Cell Biol 179(5):845–854. doi:jcb.200706040 [pii]. doi:10.1083/jcb.200706040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schober H, Ferreira H, Kalck V, Gehlen LR, Gasser SM (2009) Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination. Genes Dev 23(8):928–938. doi:23/8/928 [pii]. doi:10.1101/gad.1787509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hediger F, Neumann FR, Van Houwe G, Dubrana K, Gasser SM (2002) Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast. Curr Biol 12(24):2076–2089. doi:S0960982202013386 [pii]

    Article  CAS  PubMed  Google Scholar 

  14. Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragon L, Lisby M (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9(8):923–931. doi:10.1038/ncb1619

    Article  CAS  PubMed  Google Scholar 

  15. Mekhail K, Seebacher J, Gygi SP, Moazed D (2008) Role for perinuclear chromosome tethering in maintenance of genome stability. Nature 456(7222):667–670. doi:nature07460 [pii]. doi:10.1038/nature07460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chiolo I, Minoda A, Colmenares SU, Polyzos A, Costes SV, Karpen GH (2011) Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144(5):732–744. doi:10.1016/j.cell.2011.02.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jakob B, Splinter J, Conrad S, Voss KO, Zink D, Durante M, Lobrich M, Taucher-Scholz G (2011) DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin. Nucleic Acids Res 39(15):6489–6499. doi:10.1093/nar/gkr230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chung DK, Chan JN, Strecker J, Zhang W, Ebrahimi-Ardebili S, Lu T, Abraham KJ, Durocher D, Mekhail K (2015) Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process. Nat Commun 6:7742. doi:10.1038/ncomms8742

    Article  CAS  PubMed  Google Scholar 

  19. Chung DK, Mekhail K (2015) Repair by a molecular DNA ambulance. Oncotarget 6(23):19358–19359. doi:10.18632/oncotarget.5140

    Article  PubMed  PubMed Central  Google Scholar 

  20. Lydeard JR, Jain S, Yamaguchi M, Haber JE (2007) Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature 448(7155):820–823. doi:nature06047 [pii]. doi:10.1038/nature06047

    Article  CAS  PubMed  Google Scholar 

  21. Therizols P, Fairhead C, Cabal GG, Genovesio A, Olivo-Marin JC, Dujon B, Fabre E (2006) Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region. J Cell Biol 172(2):189–199. doi:jcb.200505159 [pii]. doi:10.1083/jcb.200505159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nagai S, Dubrana K, Tsai-Pflugfelder M, Davidson MB, Roberts TM, Brown GW, Varela E, Hediger F, Gasser SM, Krogan NJ (2008) Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322(5901):597–602. doi:322/5901/597 [pii]. doi:10.1126/science.1162790

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ricchetti M, Dujon B, Fairhead C (2003) Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast. J Mol Biol 328(4):847–862. doi:S0022283603003152 [pii]

    Article  CAS  PubMed  Google Scholar 

  24. Chapman JR, Taylor MR, Boulton SJ (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 47(4):497–510. doi:S1097-2765(12)00656-9 [pii]. doi:10.1016/j.molcel.2012.07.029

    Article  CAS  PubMed  Google Scholar 

  25. Dunham MJ, Gartenberg MR, Brown GW (2015) Methods in yeast genetics and genomics: a CSHL course manual. CSHL, New York

    Google Scholar 

  26. Lettier G, Feng Q, de Mayolo AA, Erdeniz N, Reid RJ, Lisby M, Mortensen UH, Rothstein R (2006) The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae. PLoS Genet 2(11):e194. doi:10.1371/journal.pgen.0020194

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Mekhail lab members for comments. K.M. is supported by grants from the Canadian Institutes of Health Research (CIHR), the Ontario Ministry of Research and Innovation Early Researcher Award (MRI-ERA), and the Canada Research Chair (CRC) in Spatial Genome Organization. The authors have no conflict of interest.

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Authors and Affiliations

  1. Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Ave., Toronto, ON, M5G 1M1, Canada

    Roxanne Oshidari & Karim Mekhail

  2. Canada Research Chairs Program, Faculty of Medicine, University of Toronto, 661 University Ave., Toronto, ON, M5G 1M1, Canada

    Karim Mekhail

Authors
  1. Roxanne Oshidari
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  2. Karim Mekhail
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Correspondence to Karim Mekhail .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Oshidari, R., Mekhail, K. (2018). Assays to Study Repair of Inducible DNA Double-Strand Breaks at Telomeres. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_26

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_26

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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