Production and Initial Characterization of Dad1p, a Component of the Dam1-DASH Kinetochore Complex 英文参考文献.docVIP

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Production and Initial Characterization of Dad1p, a Component of the Dam1-DASH Kinetochore Complex 英文参考文献.doc

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Production and Initial Characterization of Dad1p, a Component of the Dam1-DASH Kinetochore Complex 英文参考文献

ProductionandInitialCharacterizationofDad1p,a ComponentoftheDam1-DASHKinetochoreComplex JenniferWaldo*,MichaelScherrer DepartmentofBiology,SUNYNewPaltz,NewPaltz,NewYork,UnitedStatesofAmerica Abstract Inalldividingeukaryoticcells,themitoticspindle(composedprimarilyofmicrotubules)mustinteractwithchromosomes throughacomplexproteinassemblycalledthekinetochore.InSaccharomycescerevisiae,theDam1-DASHcomplexplaysan importantroleinpromotingattachmentbetweenthekinetochoreandthemitoticspindle.Italsoactivelyparticipatesinthe physicalseparationofsisterchromatidsinanaphase.UnderstandingthebiochemicalmechanismsusedbyDam1-DASHhas been facilitated by bacterial co-expression of the ten Dam1-DASH genes, which results in the production of a heterodecamericproteincomplexthatcanbestudiedinvitro.However,individualproteinsubunitsarenotsolublewhen expressedinE.coli,thusprecludinganalysisofthenatureoftheinteractionbetweensubunitsandanexaminationofthe assemblyofthefunctionalcomplex.Inthispaper,wedescribetheexpression,solubilization,purificationandrefoldingof Dad1p,oneoftheDam1-DASHcomplexsubunits.Inaddition,weshowthatDad1p,whenisolatedinthismannerforms dimers and/or tetramers, dependent upon protein concentration. This work provides an important tool for studying the Dam1-DASHcomplexthatwaspreviouslyunavailable,andprovidesanavenueofinvestigationforunderstandinghowthe individualheterodecamersassociatewitheachothertofacilitatechromosomesegregation. Citation:WaldoJ,ScherrerM(2008)ProductionandInitialCharacterizationofDad1p,aComponentoftheDam1-DASHKinetochoreComplex.PLoSONE3(12): e3888.doi:10.1371/journal.pone.0003888 Editor:MichaelLichten,NationalCancerInstitute,UnitedStatesofAmerica ReceivedOctober7,2008;AcceptedNovember12,2008;PublishedDecember9,2008 Copyright: ? 2008 Waldo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecr