DNA Damage, Homology-Directed Repair, and DNA Methylation 英文参考文献.docVIP

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DNA Damage, Homology-Directed Repair, and DNA Methylation 英文参考文献.doc

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DNA Damage, Homology-Directed Repair, and DNA Methylation 英文参考文献

DNADamage,Homology-DirectedRepair, andDNAMethylation Concetta Cuozzo1[,Antonio Porcellini2,3[,Tiziana Angrisano1,Annalisa Morano1,Bongyong Lee4,Alba DiPardo3, Samantha Messina3,Rodolfo Iuliano1,Alfredo Fusco1,Maria R.Santillo5,Mark T.Muller4,Lorenzo Chiariotti1, Max E.Gottesman6*,Enrico V.Avvedimento1* 1 Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita` Federico II, Naples, Italy, 2 Dipartimento di Medicina Sperimentale, Universita` ‘‘La Sapienza,’’ Rome, Italy, 3 Dipartimento di Patologia Molecolare, Istituto Neurologico Mediterraneo,Neuromed,Pozzilli,Italy,4DepartmentofMolecularBiologyandMicrobiologyandBiomolecularScienceCenter,UniversityofCentralFlorida,Orlando,Florida, UnitedStatesofAmerica,5DipartimentodiNeuroscienze,SezioneFisiologia,Universita`FedericoII,Naples,Italy,6InstituteofCancerResearch,ColumbiaUniversityMedical Center,NewYork,NewYork,UnitedStatesofAmerica ToexplorethelinkbetweenDNAdamageandgenesilencing,weinducedaDNAdouble-strandbreakinthegenomeof Helaormouseembryonicstem(ES)cellsusingI-SceIrestrictionendonuclease.TheI-SceIsitelieswithinonecopyof two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ;50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-29-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly.HalfoftheHRDNAmoleculesweredenovomethylated,principallydownstreamtothedouble-strandbreak, andhalfwereundermethylatedrelativetotheuncutDNA.Methylationoftherepairedgenewasindependentofthe methylationstatusoftheconvertingtemplate.Themethylationpatter