Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity 英文参考文献.docVIP

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Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity 英文参考文献.doc

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Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity 英文参考文献

GeneticandBiochemicalCharacterizationofHumanAP Endonuclease1MutantsDeficientinNucleotideIncision RepairActivity AuroreGelin1,ModestoRedrejo-Rodr?′guez2,JacquesLaval2,OlgaS.Fedorova3,MuratSaparbaev2, AlexanderA.Ishchenko2* 1CNRSUMR8126,Universite′ Paris-Sud,InstitutdeCance′rologie GustaveRoussy,Villejuif,France,2CNRSUMR8200Groupe?Re′paration del9ADN?,Universite′ Paris-Sud, InstitutdeCance′rologieGustaveRoussy,Villejuif,France,3InstituteofChemicalBiologyandFundamentalMedicine,SiberianBranchoftheRussianAcademyofSciences, Novosibirsk,Russia Abstract Background: Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excisionrepair(BER)andnucleotideincisionrepair(NIR)pathways.IntheBERpathway,APE1cleavesDNAatAPsitesand39- blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 59 to a number of oxidatively damagedbases.Atpresent,physiologicalrelevanceoftheNIRpathwayisfairlywellestablishedinE.coli,buthasyettobe elucidatedinhumancells. Methodology/PrincipalFinding:WeidentifiedaminoacidresiduesintheAPE1proteinthataffectitsfunctionineitherthe BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A aminoacidsubstitutionsrevealedthatallmutantsexhibitedgreatlyreducedNIRand39R59exonucleaseactivities,butwere capableofperformingBERfunctionstosomeextent.ExpressionoftheAPE1mutantsdeficientintheNIRandexonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggestingthat ourAPE1 mutantsareabletorepair APsites. Finally,thehumanNIR pathway wasfullyreconstitutedinvitrousingthepurifiedAPE1,humanflapendonuclease1,DNApolymerasebandDNAligaseI proteins,thusestablishingtheminimalsetofproteinsrequiredforafunctionalNIRpathwayinhumancells. Conclusion/Significance:Takentogether,thesedatafurthersubstantiatetheroleofNIRasadistinctandseparablefunction ofAPE1thatisesse