噬菌体的复制原理

T4噬菌体DNA的复制与调控吴俊张年辉卿人韦T4噬菌体DNA的复制与调控origin-dependentreplicationearlyininfectionrecombination-dependentreplicationatlatertimes(RDR)replicationmediatorprotein(RMP)----gp59anduvsYtheslidingclamp-----gp45Fig.2-1ModelofareplicationforkwithbacteriophageT4proteinsFig.1-1T4invitrosystemforRDRFeaturesoftheRDRpathwayFirstisthestrictrequirement,underphysiologicalconditions,foranRMPprotein(uvsY)topromotetheuvsX-catalyzedinitiationofleadingstrandsynthesisviatheDloop-formingmechanismmentionedabove.Secondistherequirementforanothermediatorprotein(gp59)toinitiatethelaggingstrandsynthesiscomponentofRDR.Thirdistheintriguingobservation,diagrammedinFig.1-1,thatthesynthesisofOkazakifragmentsalwaysoccursonthedisplacedstrandoftheDloop,andnotonthe59extensionoftheinvadingssDNA.RoleofUvsYProteininAssemblyoftheT4PresynapticFilamentUvsYhelpsuvsXdisplacegp32fromssDNA,areactionnecessaryforproperformationofthepresynapticfilament.UvsYinteractswithandstabilizesuvsX-ssDNAfilamentsaftertheyareassembled.Note:uvsXrecombinasecooperativelyboundtossDNAFig.1-2ABiochemicalmodelforuvsY-mediatedassemblyoftheT4presynapticfilament3’ssDNAtailsgeneratedduringT4origin-dependentreplicationarenaturalprimersforRDRbecausethepresenceofhomologyisguaranteedbytheterminalredundancyofT4DNA.Severalothermechanismsexistfor3’tailgeneration,includingnucleolyticresectionofDNAdouble-standbreaks(DSBs).BothDSBrepairand‘‘normal’’RDRprocessesdependontheT4gp46andgp47proteins.Theobservationofastrongprotein–proteininteractionbetweengp46/47anduvsYraisesanotherintriguingpossibility:thatnucleolyticresectionofDSBsisdirectlycoupledtotheassemblyofapresynapticfilamentontheremainingstrand.AhypotheticalmodelforthisprocessisshowninFig.1-2B.HypothesisofPresynapsisCoupledtotheNucleolyticResectionofdsDNAEndsFig.1-2BHypotheticalmodelforpresynapsiscoupledtogp46y47-catalyzedresectionofaDSBStructureofT4Gene59Helicase-LoadingProtein(gp59)T459helicase-loadingproteinisasmall,basic,almostcompletelyα-helicalproteinwhoseN-terminaldomainhasstructuralsimilaritytohighmobilitygroupfamilyproteins(HMG).Its13α-helicesaredividedintoNandCdomainsofsimilarsize.Thesingleshortβ-sheetconnectsN-terminalresidues2–4withresidues197–199neartheCterminus.Thereisanarrowgroovebetweenthetwodomainsonthe‘‘top’’oftheprotein.Thesurfaceoftheproteinisnotableforthehighdensityofbasicandhydrophobicresidues,whichmaybeimportantforitsDNAandproteininteractions(Fig.2-2).Gp59recognizesspecificstructuresratherthanspecificsequences.Itbindsandloadsthehelicaseonreplicationforksandonthree-andfour-stranded(Hollidayjunction)recombinationstructures,withoutsequencespecificity.Fig.2-2RibbondiagramsofthecrystalstructureoftheT4gene59helicase-loadingproteinshowingitsstructuralsimilaritywithHMGproteins.Fig.1-3Biochemicalmodelforgp59-ediatedhelicaseassemblyatT4replicationforkFig.1-4Enzymepartitioningmodelforstrand-specificprimingofOkazakifragmentsduringT4recombination-dependentreplicationThecoordinatedassemblyoftheDNApolymerase(gp43),theslidingclamp(gp45),andtheclamploader(gp44/62)toformthebacteriophageT4DNApolymeraseholoenzymeisamultistep.Itproceedsthrough10stepsand7conformationalchangesingp45.DNApolymeraseholoenzymeScheme1StepsintheFormationoftheT4Holoenzyme.Fig.3-1(A)X-raycrystalstructureofgp45showingtheinterdomainconnectingloopandthesubunitinterface.(B)In-planemodelofopeningofgp45showingthelocationofthemutations:V163Cinblue,S158Cingreen,andT168Cinpink,withthedonortryptophaninorange.Eachmutationisshownonlyonceforclarity.(C)Out-of-planemodelofopening.运用Fluorescenceresonanceenergytransfer(FRET)技术观测了gp45在T4DNApolymeraseholoenzyme组装过程中的动态变化，并设计了gp45的动态图。该方法的原理是：gp45的一个亚基上有一个内源色氨酸（W91)，它在能量转移中能够被检测，将它作为荧光团供体。还设计了gp45特异位置上的三个突变体（V163C,S158C,T168C),作为荧光团受体。通过计算供体和受体间的距离，得出了gp45在全酶形成中的开启和关闭模型(Fig.3-5)。FluorescenceResonanceEnergyTransferFig.3-5Molecularmodelsofgp45