Chromosoma

, Volume 117, Issue 4, pp 357–366 | Cite as

Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing

  • M. K. Bhattacharyya
  • K. M. Matthews
  • A. J. Lustig
Research Article
First Online:
Received:
Revised:
Accepted:

Abstract

Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (ΔC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. ΔC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

Keywords

Nocodazole Nuclease Activity Association Site Mre11 Complex Dysfunctional Telomere 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Notes

Acknowledgments

We thank E. B. Hoffman for critical reading of the manuscript and to Drs. John Piskur, Dan Gottschling, David Stillman, Jim Haber, and Vicki Lundblad for the gift of strains and plasmids used in this study and John Petrini for his gift of antibodies. This study was funded by NIH grant R01 GM069943 (to AJL) and the Louisiana Cancer Research Consortium (to MKB).

References

  1. Bhattacharyya MK, Lustig AJ (2006) Telomere dynamics in genome stability. Trends Biochem Sci 31:114–122PubMedCrossRefGoogle Scholar
  2. Bucholc M, Park Y, Lustig AJ (2001) Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae. Mol Cell Biol 21:6559–6573PubMedCrossRefGoogle Scholar
  3. Chamankhah M, Xiao W (1999) Formation of the yeast Mre11–Rad50–Xrs2 complex is correlated with DNA repair and telomere maintenance. Nucleic Acids Res 27:2072–2079PubMedCrossRefGoogle Scholar
  4. Cheung I, Schertzer M, Baross A, Rose AM, Lansdorp PM, Baird DM (2004) Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res 32:3383–3391PubMedCrossRefGoogle Scholar
  5. Chien CT, Bartel PL, Sternglanz R, Fields S (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A 88:9578–9582PubMedCrossRefGoogle Scholar
  6. Chien CT, Buck S, Sternglanz R, Shore D (1993) Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast. Cell 75:531–541PubMedCrossRefGoogle Scholar
  7. Craven RJ, Greenwell PW, Dominska M, Petes TD (2002) Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes. Genetics 161:493–507PubMedGoogle Scholar
  8. Diede SJ, Gottschling DE (1999) Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell 99:723–733PubMedCrossRefGoogle Scholar
  9. Diede SJ, Gottschling DE (2001) Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere. Curr Biol 11:1336–1340PubMedCrossRefGoogle Scholar
  10. Frank CJ, Hyde M, Greider CW (2006) Regulation of telomere elongation by the cyclin-dependent kinase CDK1. Mol Cell 24:423–432PubMedCrossRefGoogle Scholar
  11. Furuse M, Nagase Y, Tsubouchi H, Murakami-Murofushi K, Shibata T, Ohta K (1998) Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination. EMBO J 17:6412–6425PubMedCrossRefGoogle Scholar
  12. Ghosal G, Muniyappa K (2007) The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity. J Mol Biol 372:864–882PubMedCrossRefGoogle Scholar
  13. Grandin N, Damon C, Charbonneau M (2000) Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment. Mol Cell Biol 20:8397–8408PubMedCrossRefGoogle Scholar
  14. Grandin N, Damon C, Charbonneau M (2001) Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J 20:1173–1183PubMedCrossRefGoogle Scholar
  15. Hackett JA, Greider CW (2003) End resection initiates genomic instability in the absence of telomerase. Mol Cell Biol 23:8450–8461PubMedCrossRefGoogle Scholar
  16. Hande MP (2004) DNA repair factors and telomere-chromosome integrity in mammalian cells. Cytogenet Genome Res 104:116–122PubMedCrossRefGoogle Scholar
  17. Hemann MT, Strong MA, Hao LY, Greider CW (2001) The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107:67–77PubMedCrossRefGoogle Scholar
  18. Joseph I, Jia D, Lustig AJ (2005) Ndj1p-dependent epigenetic resetting of telomere size in yeast meiosis. Curr Biol 15:231–237PubMedCrossRefGoogle Scholar
  19. Karlseder J, Smogorzewska A, de Lange T (2002) Senescence induced by altered telomere state, not telomere loss. Science 295:2446–2449PubMedCrossRefGoogle Scholar
  20. Krogh BO, Llorente B, Lam A, Symington LS (2005) Mutations in Mre11 phosphoesterase motif I that impair Saccharomyces cerevisiae Mre11–Rad50–Xrs2 complex stability in addition to nuclease activity. Genetics 171:1561–1570PubMedCrossRefGoogle Scholar
  21. Kyrion G, Liu K, Liu C, Lustig AJ (1993) RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae. Genes Dev 7:1146–1159PubMedCrossRefGoogle Scholar
  22. Lee SE, Bressan DA, Petrini JH, Haber JE (2002) Complementation between N-terminal Saccharomyces cerevisiae mre11 alleles in DNA repair and telomere length maintenance. DNA Repair (Amst) 1:27–40CrossRefGoogle Scholar
  23. Lewis LK, Karthikeyan G, Westmoreland JW, Resnick MA (2002) Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase). Genetics 160:49–62PubMedGoogle Scholar
  24. Lydall D (2003) Hiding at the ends of yeast chromosomes: telomeres, nucleases and checkpoint pathways. J Cell Sci 116:4057–4065PubMedCrossRefGoogle Scholar
  25. Maringele L, Lydall D (2002) EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants. Genes Dev 16:1919–1933PubMedCrossRefGoogle Scholar
  26. Maringele L, Lydall D (2004) EXO1 plays a role in generating type I and type II survivors in budding yeast. Genetics 166:1641–1649PubMedCrossRefGoogle Scholar
  27. Martens UM, Chavez EA, Poon SS, Schmoor C, Lansdorp PM (2000) Accumulation of short telomeres in human fibroblasts prior to replicative senescence. Exp Cell Res 256:291–299PubMedCrossRefGoogle Scholar
  28. Moreau S, Ferguson JR, Symington LS (1999) The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance. Mol Cell Biol 19:556–566PubMedGoogle Scholar
  29. Nakada D, Matsumoto K, Sugimoto K (2003) ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 17:1957–1962PubMedCrossRefGoogle Scholar
  30. Nugent CI, Hughes TR, Lue NF, Lundblad V (1996) Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274:249–252PubMedCrossRefGoogle Scholar
  31. Pabla R, Pawar V, Zhang H, Siede W (2006) Characterization of checkpoint responses to DNA damage in Saccharomyces cerevisiae: basic protocols. Methods Enzymol 409:101–117PubMedCrossRefGoogle Scholar
  32. Park Y, Hanish J, Lustig AJ (1998) Sir3p domains involved in the initiation of telomeric silencing in Saccharomyces cerevisiae. Genetics 150:977–986PubMedGoogle Scholar
  33. Sandell LL, Zakian VA (1993) Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75:729–739PubMedCrossRefGoogle Scholar
  34. Shi QM, Wang YM, Zheng XD, Teck Ho Lee R, Wang Y (2006) Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans Mol Biol Cell 18:815–826Google Scholar
  35. Shima H, Suzuki M, Shinohara M (2005) Isolation and characterization of novel xrs2 mutations in Saccharomyces cerevisiae. Genetics 170:71–85PubMedCrossRefGoogle Scholar
  36. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  37. Steiner BR, Hidaka K, Futcher B (1996) Association of the Est1 protein with telomerase activity in yeast. Proc Natl Acad Sci U S A 93:2817–2821PubMedCrossRefGoogle Scholar
  38. Taggart AK, Teng SC, Zakian VA (2002) Est1p as a cell cycle-regulated activator of telomere-bound telomerase. Science 297:1023–1026PubMedCrossRefGoogle Scholar
  39. Takata H, Tanaka Y, Matsuura A (2005) Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication proteins in Saccharomyces cerevisiae. Mol Cell 17:573–583PubMedCrossRefGoogle Scholar
  40. Tauchi H, Kobayashi J, Morishima K, van Gent DC, Shiraishi T, Verkaik NS, vanHeems D, Ito E, Nakamura A, Sonoda E, Takata M, Takeda S, Matsuura S, Komatsu K (2002) Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420:93–98PubMedCrossRefGoogle Scholar
  41. Tran PT, Erdeniz N, Symington LS, Liskay RM (2004) EXO1-A multi-tasking eukaryotic nuclease. DNA Repair (Amst) 3:1549–1559CrossRefGoogle Scholar
  42. Trujillo KM, Sung P (2001) DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex. J Biol Chem 276:35458–35464PubMedCrossRefGoogle Scholar
  43. Tseng SF, Lin JJ, Teng SC (2006) The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation. Nucleic Acids Res 34:6327–6336Google Scholar
  44. Usui T, Ohta T, Oshiumi H, Tomizawa J, Ogawa H, Ogawa T (1998) Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95:705–716PubMedCrossRefGoogle Scholar
  45. Williams B, Bhattacharyya MK, Lustig AJ (2005) Mre11p nuclease activity is dispensable for telomeric rapid deletion. DNA Repair (Amst) 4:994–1005CrossRefGoogle Scholar
  46. Wiltzius JJ, Hohl M, Fleming JC, Petrini JH (2005) The Rad50 hook domain is a critical determinant of Mre11 complex functions. Nat Struct Mol Biol 12:403–407PubMedCrossRefGoogle Scholar
  47. You Z, Chahwan C, Bailis J, Hunter T, Russell P (2005) ATM activation and its recruitment to damaged DNA require binding to the C Terminus of Nbs1. Mol Cell Biol 25:5363–5379PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • M. K. Bhattacharyya
    • 1
  • K. M. Matthews
    • 1
  • A. J. Lustig
    • 1
  1. 1.Department of BiochemistryTulane University Health Sciences CenterNew OrleansUSA

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