Sensitivity of Yeast Strains with Long G-Tails to Levels of Telomere-Bound Telomerase 英文参考文献.docVIP

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Sensitivity of Yeast Strains with Long G-Tails to Levels of Telomere-Bound Telomerase 英文参考文献.doc

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Sensitivity of Yeast Strains with Long G-Tails to Levels of Telomere-Bound Telomerase 英文参考文献

SensitivityofYeastStrainswithLong G-TailstoLevelsofTelomere-Bound Telomerase Leticia R.Vega1[¤,Jane A.Phillips1[,Brian R.Thornton2,Jennifer A.Benanti2,Mutiat T.Onigbanjo1, David P.Toczyski2,Virginia A.Zakian1* 1DepartmentofMolecularBiology,PrincetonUniversity,Princeton,NewJersey,UnitedStatesofAmerica, 2CancerResearchInstitute,DepartmentofBiochemistryand Biophysics,UniversityofCalifornia,SanFranciscoCalifornia,UnitedStatesofAmerica The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughoutmostofthecellcycle,withpeaksofassociationinbothG1phase(whentelomeraseisnotactive)andlate S/G2phase (when telomeraseis active). The G1association ofEst2p requiresa specific interaction betweenKu and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundanceinG1phasewasanaphase-promotingcomplexdependent.Thus,endogenousPif1pisunlikelytoactonG1 boundEst2p.OverexpressionofPif1pfromanon-cellcycle-regulatedpromoterdramaticallyreducedviabilityinfive strainswithimpairedendprotection(cdc13–1,yku80D,yku70D,yku80–1,andyku80–4),allofwhichhavelongersingle- strandG-tailsthanwild-typecells.ThisreducedviabilitywassuppressedbydeletingtheEXO1gene,whichencodesa nucleasethatactsatcompromisedtelomeres,suggestingthattheremovaloftelomerasebyPif1pexposedtelomeres tofurtherC-stranddegradation.Consistentwiththisinterpretation,depletionofPif1p,whichincreasestheamountof telomere-bound telomerase, suppressed the temperature sensitivity of yku70D and cdc13–1 cells. Furthermore, eliminatingthepathwaythatrecruitsEst2ptotelomeresinG1phaseinacdc13–1strainalsor