DNA復(fù)制起始蛋白Mcm6-Cdt1相互作用的研究.pdf

1、華東師范大學(xué)博士學(xué)位論文DNA復(fù)制起始蛋白Mcm6Cdt1相互作用的研究姓名：魏準(zhǔn)申請學(xué)位級別：博士專業(yè)：無線電物理指導(dǎo)教師：朱廣20100401AbstractInalleukaryoticcells，initiationofgenomicDNAreplicationistightlycontrolledtoensurethatDNAreplicationOCCurSonlyonceineachcellcycleDNAreplicat

2、ioninitiationisthecriticalstepduringthewholeDNAreplicationprocess，initiationstepisstrictlycontrolledbytheformationofprereplicationcomplex(pre—RC)whichiscomposedofseveralimportantproteincomplexesandproteinsincludingthemin

3、ichromosomemaintenanceMCMcomplexTheMCMcomplexiscomposedofsixevolutionallyconservedproteinsMcm2Mcm7，anditisbelievedtobethereplicativehelicaseineukaryoticspecies，anditplaysessentialrolesintheinitiationandelongationphasesof

4、DNAreplicationDuringlateMandearlyG1，theMcm2—7complexisloadedontochromatintoformpre—RCinaCdtldependentmannerMcm6isoneofthesixsubunitoftheMcm27complex，anditcontainsthehighlyconservedMCMdomainiandoneuncharacterizedCterminal

5、domainInthisstudywedemonstratethatthepreviouslyuncharacterizedC—terminaldomainofhumanMcm6istheCdtlbindingdomain(CBD)Withtheuseofhi曲resolutionnuclearmagneticresonance(NMR)technique，wesolvedthe3DstructureofCBDThestructureo

6、fCBDexhibitsatypical“winged—helix’’foldthatisgenerallyinvolvedinproteinnucleicacidinteractionNevertheless，theCBDfailedtointeract塒thDNAinOurstudiesindicatingthatitisspecificforproteinproteininteractionAlso，wemappedtheMcm6

7、bindingdomain(MBD)ofCdtlBasedonNMRtitrationexperimentandGSTPull—downassayweidentifiedE757andL766onCBDarethecriticalresiduesfortheinteractionwithCdtlThefundamentalroleofthesetwesiduesinMcm6Cdtiinteractionwasfurtherconfirm

8、edwithsubcellularCO—localizationassayBasedontheCBDstructure，wehavedeterminedthehi曲resolutionstructureoftheCBD—MBDcomplexAccumulatingstudiesindicatethatCdtlmediatethechromatinloadingofMcm2—7complex；howeverthedetailedmolec

9、ularmechanismremainselusiveInordertoelucidatethedetailedroleplayedbyCdtlinthepre—RCassemblywearecurrentlyutilizingXenopuseggextractsystemtoinvestigatethebiologicalconsequenceofMcm6一CdtlinteractionBasedonourpreviousbioche

10、micalandstructuralstudies，wehavegotquiteencouragingresultsinthefunctionalinvestigationsWespeculatethatCdtldirectsMcrn27complexchromatinloadingtoaccomplishpre—RCformationthroughthedirectinteractionbetweentheMBDofCdtlandCB