參與原核生物DNA復(fù)制的酶類和蛋白質(zhì)EnzymesandProteins教案資料

參與原核生物DNA復(fù)制的酶類和蛋白質(zhì)EnzymesandProteinsInvolvedinDNAReplicationinProkaryotes

高方遠(yuǎn)馬欣榮康海岐DNAreplication(bacteria)InitiationElongationTerminationDaughterDNApartition*theoriginofreplicationisdefined

*replicationbubble.*Replicationfork*Unidirectionalorbidirectional.Originstermination參與DNA復(fù)制的酶類1、DNA聚合酶2、DNA引發(fā)酶（DNAprimase）3、DNA連接酶4、與DNA幾何學(xué)性質(zhì)相關(guān)的酶

IpolAmajorrepairenzymeIIpolBminorrepairenzymeIIIpolCreplicase

IVdinBSOSrepairVumuC、DSOSrepairDNApolymerasesinE.coliDNAPolymeraseIDNApolymerase3’5’exonuclease5’3’exonucleaseFigure13.8ThecatalyticdomainofaDNApolymerasehasaDNA-bindingcleftcreatedbythreesubdomains.Theactivesiteisinthepalm.Proofreadingisprovidedbyaseparateactivesiteinanexonucleasedomain.Figure13.7CrystalstructureofphageT7DNApolymerasehasarighthandstructure.DNAliesacrossthepalmandisheldbythefingersandthumb.PhotographkindlyprovidedbyCharlesRichardsonandTomEllenberger.Figure13.5Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.DNAPolymeraseISubunitcompositionofE.coliDNApolymeraseIIIholoenzyme

subunitmolecularmassfunctionsubassemblies(KDa)

α129.9DNApolymeraseε27.53’5’exonuclease

coreθ8.6stimulatesexonucleaseι71.1dimerizescorePolIII’bindsγcomplexγ47.5bindsATPδ38.7bindstoβPolIIIδ’36.9bindstoγandδγcomplexχ16.6bindstoSSBDNA-dependentψ15.2bindstoχandγATPaseβ40.6slidingclampE.coliPolIIIBeta-subunitFigure13.18DNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrands.Fig.1.

ModelofSOStranslesionreplicationbyDNApolymeraseV.

ThetwoDNAstrandsareshownasgreenlines,andthereplication-blockinglesionisrepresentedbytheredrectangle.ThethreemajorstepsinTLRarepre-initiation(2),inwhichtheRecAnucleoproteinfilamentsassembles;initiation(3

and4),whichinvolvesbindingofpolVtotheprimer-templateandloadingofthe

subunitclamp;andlesionbypassbypolVholoenzyme(5).SSBissuggestedtohelpindisplacingRecAfromDNAbothattheinitiationandlesionbypasssteps.

E.coliDNApolymeraseIV

（dinBgene）

*dinB基因的表達(dá)需要DNA損傷誘導(dǎo)*與UmuC、UmuD同屬Y家族DNA聚合酶*E.coliDNApolymeraseIV無校正功能，易于延長一些凸出的引物或模板結(jié)構(gòu)。2、DNA引發(fā)酶（DNAprimase）

UsehostRNApolymeraseasprimase(M13)primosomeprimase(dnaGprotein)(E.coli)otherproteins

фX174:onlyprimase,withouttheotherproteins

Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.Adenovirusterminalproteinbindstothe5

endofDNAandprovidesaC-OHendtoprimesynthesisofanewDNAstrand.AprimerterminusisgeneratedwithinduplexDNA.Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.DNAPolymeraseI與DNA幾何學(xué)性質(zhì)相關(guān)的酶解旋酶（Helicase）拓?fù)洚悩?gòu)酶（Topoisomerases）解旋酶（Helicase）至少4種helicases*rephelicase*DNAhelicaseII*DNAhelicaseIII

*dnaBProtein:在E.coliDNA復(fù)制中解開DNA雙鏈拓?fù)洚悩?gòu)酶（Topoisomerases）拓?fù)洚悩?gòu)酶I(topAgene)actonhighlynegativelysupercoiledDNAstabilizesingle-strandedregionsFigure14.16BacterialtypeItopoisomerasesrecognizepartiallyunwoundsegmentsofDNAandpassonestrandthroughabreakmadeintheother.拓?fù)洚悩?gòu)酶II

TypeIItopoisomerasesgenerallyrelaxbothnegativeandpositivesupercoils.ThereactionrequiresATPFigure14.17TypeIItopoisomerasescanpassaduplexDNAthroughadouble-strandbreakinanotherduplex.

拓?fù)洚悩?gòu)酶IV與子代DNA分子的分開有關(guān)參與DNA復(fù)制的蛋白質(zhì)1、參與復(fù)制起始的蛋白質(zhì)因子Originalcomplx:DnaA、DnaB、DnaC、DnaG、HUandSSB

Theminimaloriginisdefinedbythedistancebetweentheoutsidemembersofthe13-merand9-merrepeats

Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,leadingtotheseparationofDNAstrands.methylationattheoriginAmembrane-boundinhibitorbindstohemimethylatedDNAattheorigin,andmayfunctionbypreventingthebindingofDnaA.ItisreleasedwhentheDNAisremethylated.SeqAThecomplexatoriCcanbedetectedbyelectronmicroscopy.

AntibodiesofdnaAproteinHUTheproteinHUisageneralDNA-bindingproteininE.coli.Itspresenceisnotabsolutelyrequiredtoinitiatereplicationinvitro,butitstimulatesthereaction.HUhasthecapacitytobendDNA,andislikelytobeinvolvedinsomegeneralstructuralcapacity.2、參與復(fù)制延伸的蛋白質(zhì)因子(DnaG)(DnaB)2、參與復(fù)制終止的蛋白質(zhì)因子TusHowdothedaughterDNAsbecomedisentangled?

與真核生物不同，細(xì)菌的DNA復(fù)制、染色體重新折疊以及姊妹染色體的分開是同時發(fā)生的。細(xì)菌中姊妹染色體的分開的機(jī)制與真核生物不同。細(xì)菌染色體的DNA分子本身卷入了分開機(jī)制。細(xì)菌的多復(fù)制叉復(fù)制（multiplereplication）與真核生物的復(fù)制方式不同。*多拷貝的oriC*只有一個終止序列Asimplifiedmodelofthebacterialcellcycle.Themodelissimplifiedtoignoremultiforkreplication.SMC類似物

Amodelofacircularchromosomethatisundergoingmultiforkreplicationinarod-shapedbacterium.復(fù)制起點(diǎn)及終點(diǎn)在細(xì)胞中的位置1、膜片段中有復(fù)制起始區(qū)與終止區(qū)的富集推斷錨定蛋白在定位中的作用。SeqA2、位于中間位置復(fù)制工廠的動力作用多蛋白復(fù)合體：polymerase,helicaseandaccociatedproteins

特殊蛋白質(zhì)（PolC-GFP、SeqA)的定位；H3同位素標(biāo)記；pullDNAtemplateduplicatedreleaseDNAoutwardduringreplicationTheextrusion-capturemodelforbacterialchromosomepartitioning.3、與染色體組織（organization）、緊結(jié)(compaction)和超螺旋(helicase)有關(guān)的蛋白質(zhì)作用SMCMukB

toorganizethechromosomeintoahigherorderstructurebyconstrainingsupercoils.(cause)

PartitioningMotorprotein

(altered)Chromosomepartitioning(consequence)HU;Hbsu;Terminus-specificchromosomeparti