Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR 英文参考文献.docVIP

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Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR 英文参考文献.doc

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Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR 英文参考文献

ProliferatingCellNuclearAntigen(PCNA)Interactionsin SolutionStudiedbyNMR AlfredoDeBiasio1.,Ramo′nCampos-Olivas2*.,RicardoSa′nchez1,JorgeP.Lo′pez-Alonso2, DavidPantoja-Uceda3,NekaneMerino1,MaiderVillate1,JoseM.Martin-Garcia4,FranciscoCastillo4, IreneLuque4,FranciscoJ.Blanco1,5* 1StructuralBiologyUnit,CICbioGUNE,Derio,Spain,2SpectroscopyandNMRUnit,CentroNacionaldeInvestigacionesOncolo′gicas,Madrid,Spain,3InstitutodeQu?′mica F?′sica Rocasolano, Consejo Superior de Investigaciones Cient?′ficas, Madrid, Spain, 4Department of Physical Chemistry and Institute of Biotechnology, University of Granada,Granada,Spain,5IKERBASQUE,BasqueFoundationforScience,Bilbao,Spain Abstract PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86kDa by the symmetric associationofthreeidenticalprotomers.TheringencirclestheDNAandactsasadockingplatformforotherproteins,most ofthemcontainingthePCNAInteractionProteinsequence(PIP-box).WehaveusedNMRtocharacterizetheinteractionsof PCNAwithseveralotherproteinsandfragmentsinsolution.ThebindingofthePIP-boxpeptideofthecellcycleinhibitor p21toPCNAisconsistentwiththecrystalstructureofthecomplex.Ashorterp21peptidebindswithreducedaffinitybut retainsmostofthemolecularrecognitiondeterminants.Howeverthebindingofthecorrespondingpeptideofthetumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reducetheaffinityforPCNA,incontrastwithaproposedlessstringentPIP-boxsequencerequirement.Wecouldnotdetect anybindingbetweenPCNAandtheMCL-1ortheCDK2protein,reportedtointeractwithPCNAinbiochemicalassays.This suggeststhattheydonotbinddirectlytoPCNA,ortheydobutveryweakly,withadditionalunidentifiedfactorsstabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, includingtheinterdomainconnectorloop(IDCL)andtheC-terminus,bothoftheminvolvedintheinteractionwiththePIP- box.Ourworkprovidesthebasisforhigh