Base Flipping in Tn10 Transposition An Active Flip and Capture Mechanism 英文参考文献.docVIP

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Base Flipping in Tn10 Transposition An Active Flip and Capture Mechanism 英文参考文献.doc

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Base Flipping in Tn10 Transposition An Active Flip and Capture Mechanism 英文参考文献

BaseFlippinginTn10Transposition:AnActiveFlipand CaptureMechanism JulienBischerour¤,RonaldChalmers* UniversityofNottingham,SchoolofBiomedicalSciences,TheMedicalSchool,QueensMedicalCentre(QMC),Nottingham,UnitedKingdom Abstract The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposonends.ThisisachievedusingahairpinintermediatethatrequirestheDNAtochangeconformationduringthe reaction.InTn5thesechangesarecontrolledinpartbyaflippednucleosidethatisstackedonatryptophanresidueina hydrophobicpocketofthetransposase.HerewehaveinvestigatedthebaseflippingmechanisminTn10transposition.Asin Tn5transposition,wefindthatbaseflippingtakesplaceafterthefirstnickandisrequiredforefficienthairpinformationand resolution.Experimentswithanabasicsubstrateshowthattheroleofbaseflippinginhairpinformationistoremovethe basefromtheDNAhelix.Specificinteractionsbetweentheflippedbaseandthestackingtryptophanresiduearerequired for hairpin resolution later in the reaction. We showthat base flipping in Tn10 transposition is not apassive reactionin whichaspontaneouslyflippedbaseiscapturedandretainedbytheprotein.Rather,itisdriveninpartbyamethionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transpositionissimilartothatinTn5transposition. Citation: Bischerour J, Chalmers R (2009) Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism. PLoS ONE 4(7): e6201. doi:10.1371/ journal.pone.0006201 Editor:Anja-KatrinBielinsky,UniversityofMinnesota,UnitedStatesofAmerica ReceivedMarch25,2009;AcceptedMay25,2009;PublishedJuly10,2009 Copyright:?2009Bischerour,Chalmers.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwasfundedbyagrantfromTheWellcomeTrust.Thefundershadnoroleinstudydesign,datacol