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The Telomerase Subunit Est3 Binds Telomeres in a Cell Cycle– and Est1–Dependent Manner and Interacts Directly with Est1


Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.


Vyšlo v časopise: The Telomerase Subunit Est3 Binds Telomeres in a Cell Cycle– and Est1–Dependent Manner and Interacts Directly with Est1. PLoS Genet 7(5): e32767. doi:10.1371/journal.pgen.1002060
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002060

Souhrn

Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.


Zdroje

1. GertonJLHawleyRS 2005 Homologous chromosome interactions in meiosis: diversity amidst conservation. Nat Rev Genet 6 477 487

2. VegaLMateyakMZakianV 2003 Getting to the end: telomerase access in yeast and humans. Nat Rev Mol Cell Biol 4 948 959

3. de LangeT 2005 Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19 2100 2110

4. LarriveeMLeBelCWellingerRJ 2004 The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex. Genes Dev 18 1391 1396

5. LingnerJHughesTRShevchenkoAMannMLundbladV 1997 Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276 561 567

6. SingerMSGottschlingDE 1994 TLC1, the template RNA component of the Saccharomyces cerevisiae telomerase. Science 266 404 409

7. LundbladVSzostakJW 1989 A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57 633 643

8. LendvayTSMorrisDKSahJBalasubramanianBLundbladV 1996 Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. Genetics 144 1399 1412

9. HughesTREvansSKWeilbaecherRGLundbladV 2000 The Est3 protein is a subunit of yeast telomerase. Curr Biol 10 809 812

10. BournsBDAlexanderMKSmithAMZakianVA 1998 Sir proteins, Rif proteins and Cdc13p bind Saccharomyces telomeres in vivo. Mol Cell Biol 18 5600 5608

11. TsukamotoYTaggartAKPZakianVA 2001 The role of the Mre11-Rad50-Xrs2 complex in telomerase-mediated lengthening of Saccharomyces cerevisiae telomeres. Curr Biol 11 1328 1335

12. NugentCIHughesTRLueNFLundbladV 1996 Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274 249 252

13. GarvikBCarsonMHartwellL 1995 Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol 15 6128 6138

14. EvansSKLundbladV 1999 Est1 and Cdc13 as comediators of telomerase access. Science 286 117 120

15. BianchiANegriniSShoreD 2004 Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1. Mol Cell 16 139 146

16. McCarrollRMFangmanWL 1988 Time of replication of yeast centromeres and telomeres. Cell 54 505 513

17. WellingerRJWolfAJZakianVA 1993 Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a yeast linear plasmid. Mol Cell Biol 13 4057 4065

18. WellingerRJWolfAJZakianVA 1993 Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase. Cell 72 51 60

19. BonettiDMartinaMClericiMLucchiniGLongheseMP 2009 Multiple pathways regulate 3′ overhang generation at S. cerevisiae telomeres. Mol Cell 35 70 81

20. DiedeSJGottschlingDE 1999 Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell 99 723 733

21. MarcandSBrevetVMannCGilsonE 2000 Cell cycle restriction of telomere elongation. Curr Biol 10 487 490

22. TaggartAKPTengS-CZakianVA 2002 Est1p as a cell cycle-regulated activator of telomere-bound telomerase. Science 297 1023 1026

23. FisherTTaggartAZakianV 2004 Cell cycle-dependent regulation of yeast telomerase by Ku. Nat Struct Mol Biol 11 1198 1205

24. ChanABouleJBZakianVA 2008 Two pathways recruit telomerase to Saccharomyces cerevisiae telomeres. PLoS Genet 4 e1000236 doi:10.1371/journal.pgen.1000236

25. OsterhageJLTalleyJMFriedmanKL 2006 Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae. Nat Struct Mol Biol 13 720 728

26. QiHZakianVA 2000 The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein. Genes Dev 14 1777 1788

27. PennockEBuckleyKLundbladV 2001 Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104 387 396

28. LinJ-JZakianVA 1995 An in vitro assay for Saccharomyces telomerase requires EST1. Cell 81 1127 1135

29. DeZwaanDCFreemanBC 2009 The conserved Est1 protein stimulates telomerase DNA extension activity. Proc Natl Acad Sci 106 17337 17342

30. HsuMYuEYSinghSMLueNF 2007 Mutual dependence of Candida albicans Est1p and Est3p in telomerase assembly and activation. Eukaryot Cell 6 1330 1338

31. LeeJMandellEKRaoTWuttkeDSLundbladV 2010 Investigating the role of the Est3 protein in yeast telomere replication. Nucleic Acids Res 38 2279 2290

32. LeeJMandellEKTuceyTMMorrisDKLundbladV 2008 The Est3 protein associates with yeast telomerase through an OB-fold domain. Nat Struct Mol Biol 15 990 997

33. YuEYWangFLeiMLueNF 2008 A proposed OB-fold with a protein-interaction surface in Candida albicans telomerase protein Est3. Nat Struct Mol Biol 15 985 989

34. HockemeyerDPalmWElseTDanielsJPTakaiKK 2007 Telomere protection by mammalian Pot1 requires interaction with Tpp1. Nat Struct Mol Biol 14 754 761

35. WangFPodellERZaugAJYangYBaciuP 2007 The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature 445 506 510

36. XinHLiuDWanMSafariAKimH 2007 TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase. Nature 445 559 562

37. SabourinMTuzonCFisherTZakianV 2007 A flexible protein linker improves the function of epitope-tagged proteins in Saccharomyces cerevisiae. Yeast 24 39 45

38. MaierTDrapalNThanbichlerMBockA 1998 Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. Anal Biochem 259 68 73

39. GoudsouzianLTuzonCZakianVA 2006 S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association. Mol Cell 24 603 610

40. SabourinMTuzonCVAZ 2007 Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Mol Cell 27 550 561

41. VodenicharovMWellingerRJ 2006 DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase. Mol Cell 24 127 137

42. FrankCJHydeMGreiderCW 2006 Regulation of telomere elongation by the cyclin-dependent kinase CDK1. Mol Cell 24 423 432

43. LiSMakovetsSMatsuguchiTBlethrowJDShokatKM 2009 Cdk1-dependent phosphorylation of Cdc13 coordinates telomere elongation during cell-cycle progression. Cell 136 50 61

44. TsengSFShenZJTsaiHJLinYHTengSC 2009 Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13. Nucleic Acids Res 37 3602 3611

45. KinoshitaETakahashiMTakedaHShiroMKoikeT 2004 Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex. Dalton Trans 1189 1193

46. BianchiAShoreD 2007 Increased association of telomerase with short telomeres in yeast. Genes Dev 21 1726 1730

47. ArnericMLingnerJ 2007 Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast. EMBO Rep 8 1080 1085

48. HectorRShtofmanRRayAChenBNyunT 2007 Tel1p preferentially associates with short telomeres to stimulate their elongation. Mol Cell 27 851 858

49. LueNF 2010 Plasticity of telomere maintenance mechanisms in yeast. Trends Biochem Sci 35 8 17

50. KelleherCKurthILingnerJ 2005 Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol Cell Biol 25 808 818

51. ZappullaDCRobertsJNGoodrichKJCechTRWuttkeDS 2009 Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Res 37 354 367

52. FriedmanKLHeitJJLongDMCechTR 2003 N-terminal domain of yeast telomerase reverse transcriptase: recruitment of Est3p to the telomerase complex. Mol Biol Cell 14 1 13

53. GhaemmaghamiSHuhWKBowerKHowsonRWBelleA 2003 Global analysis of protein expression in yeast. Nature 425 737 741

54. MozdyADCechTR 2006 Low abundance of telomerase in yeast: implications for telomerase haploinsufficiency. RNA 12 1721 1737

55. YangCPChenYBMengFLZhouJQ 2006 Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo. Nucl Acids Res 34 407 416

56. ZhangMLTongXJFuXHZhouBOWangJ 2010 Yeast telomerase subunit Est1p has guanine quadruplex-promoting activity that is required for telomere elongation. Nat Struct Mol Biol 17 202 209

57. ZhouJHidakaKFutcherB 2000 The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA. Mol Cell Biol 20 1947 1955

58. SikorskiRSHieterP 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122 19 27

59. MorrisDKLundbladV 1997 Programmed translational frameshifting in a gene required for yeast telomere replication. Curr Biol 7 969 976

60. FoianiMMariniFGambaDLucchiniGPlevaniP 1994 The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication. Mol Cell Biol 14 923 933

61. KushnirovVV 2000 Rapid and reliable protein extraction from yeast. Yeast 16 857 860

62. WanSCapassoHWalworthNC 1999 The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast. Yeast 15 821 828

63. RungeKWZakianVA 1989 Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol Cell Biol 9 1488 1497

64. McGeeJPhillipsJChanASabourinMPaeschkeK 2010 Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair. Nature Struct Molec Biol 17 1438 1445

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