Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis 英文参考文献.docVIP

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Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis 英文参考文献.doc

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Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis 英文参考文献

DissectionofStructureandFunctionoftheN-Terminal DomainofMouseDNMT1UsingRegionalFrame-Shift Mutagenesis LeonardoD’Aiuto1,MarcoMarzulli1,K.NagaMohan1,EwaBorowczyk1,FedericaSaporiti3 ,Andrew VanDemark2,J.RichardChaillet1* 1DepartmentofMicrobiologyandMolecularGenetics,UniversityofPittsburgh,Pittsburgh,Pennsylvania,UnitedStatesofAmerica,2DepartmentofBiologicalSciences, UniversityofPittsburgh,Pittsburgh,Pennsylvania,UnitedStatesofAmerica,3DepartmentofNeurology,UniversityofPittsburgh,Pittsburgh,Pennsylvania,UnitedStates ofAmerica Abstract DeletionanalysisofmouseDNMT1,theprimarymaintenancemethyltransferaseinmammals,showedthatmostoftheN- terminal regulatory domain (amino acid residues 412-1112) is required for its enzymatic activity. Although analysis of deletionmutantshelpstoidentifyregionsofaproteinsequencerequiredforaparticularactivity,aminoaciddeletionscan have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed RegionalFrame-shiftMutagenesis(RFM)asanewapproachtoidentifyportionsrequiredforthemethyltransferaseactivity ofDNMT1withintheN-terminal89-905aminoacids.Inthismethod,ashortstretchofaminoacidsinthewild-typeprotein isconvertedtoadifferentaminoacidsequence.Theresultantmutantproteinretainsthesameaminoacidlengthasthe wildtype,therebyreducingphysicalconstrainsonnormalfoldingofthemutantprotein.UsingRFM,weidentifiedthree smallregionsintheamino-terminalone-thirdoftheproteinthatareessentialforDNMT1function.Twooftheseregions (aminoacids124-160and341-368)borderalargedisorderedregionthatregulatesmaintenancemethylationactivity.This organizationofDNMT1’saminoterminussuggeststhatthebordersdefinethepositionofthedisorderedregionwithinthe DNMT1protein,whichinturnallowsforitsproperfunction. Citation:D’AiutoL,MarzulliM,MohanKN,BorowczykE,SaporitiF,etal.(2010)DissectionofStructureandFunctionoftheN-TerminalDomain