参与原核生物DNA复制的酶类和蛋白质EnzymesandProteins.ppt

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1、参与原核生物DNA复制的酶类和蛋白质EnzymesandProteins Still waters run deep.流静水深流静水深,人静心深人静心深 Where there is life,there is hope。有生命必有希望。有生命必有希望DNAreplication(bacteria)InitiationElongationTerminationDaughterDNApartition*theoriginofreplicationisdefined*replicationbubble.*Replicationfork*Unidirectionalorbidirectional.O

2、riginsFigure13.9Theleadingstrandissynthesizedcontinuouslywhilethelaggingstrandissynthesizeddiscontinuously.Elongation(semidiscontinuous)termination参与参与DNA复制的酶类复制的酶类1 1、DNADNA聚合酶聚合酶2 2、DNADNA引发酶（引发酶（DNA primaseDNA primase）3 3、DNADNA连接酶连接酶4 4、与、与DNADNA几何学性质相关的酶几何学性质相关的酶 IpolAmajorrepairenzymeIIpolBmin

3、orrepairenzymeIIIpolCreplicaseIVdinBSOSrepairVumuC、DSOSrepairDNApolymerasesinE.coliDNAPolymeraseIDNApolymerase35exonuclease53exonucleaseFigure 13.8 The catalytic domain of a DNA polymerase has a DNA-binding cleft created by three subdomains.The active site is in the palm.Proofreading is provided by

4、a separate active site in an exonuclease domain.Figure13.7CrystalstructureofphageT7DNApolymerasehasarighthandstructure.DNAliesacrossthepalmandisheldbythefingersandthumb.PhotographkindlyprovidedbyCharlesRichardsonandTomEllenberger.Figure13.5Nicktranslationreplacespartofapre-existingstrandofduplexDNAw

5、ithnewlysynthesizedmaterial.DNAPolymeraseISubunitcompositionofE.coli DNApolymeraseIIIholoenzymesubunitmolecularmassfunctionsubassemblies(KDa)129.9DNApolymerase27.535exonucleasecore8.6stimulatesexonuclease71.1dimerizescorePolIIIbindscomplex47.5bindsATP38.7bindstoPolIII36.9bindstoandcomplex16.6bindsto

6、SSBDNA-dependent15.2bindstoandATPase40.6slidingclampE.coliPolIIIBeta-subunitFigure13.18DNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrands.Fig.1.ModelofSOStranslesionreplicationbyDNApolymeraseV.ThetwoDNAstrandsareshownasgreen lines,andthereplica

7、tion-blockinglesionisrepresentedbythered rectangle.ThethreemajorstepsinTLRarepre-initiation(2),inwhichtheRecAnucleoproteinfilamentsassembles;initiation(3and4),whichinvolvesbindingofpolVtotheprimer-templateandloadingofthesubunitclamp;andlesion bypassbypolVholoenzyme(5).SSBissuggestedtohelpindisplacin

8、gRecAfromDNAbothattheinitiationandlesionbypasssteps.E.coli DNA polymerase IV（dinBgene）*dinB dinB 基因的表达需要基因的表达需要 DNA DNA损伤诱导损伤诱导 *与与UmuCUmuC、UmuD UmuD同属同属Y Y 家族家族DNADNA聚合酶聚合酶 *E.coliDNApolymeraseIV无校正功能，易于延长一些凸无校正功能，易于延长一些凸出的引物或模板结构。出的引物或模板结构。2 2、DNADNA引发酶（引发酶（DNA primaseDNA primase）UsehostRNApolymer

9、aseasprimase(M13)primosomeprimase(dnaGprotein)(E.coli)otherproteinsX174:onlyprimase,withouttheotherproteinsInitiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.Adenovirusterminalproteinbindstothe5 endofDNAandprovidesaC-OHendtoprimesynth

10、esisofanewDNAstrand.AprimerterminusisgeneratedwithinduplexDNA.Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.DNAPolymeraseI与与DNADNA几何学性质相关的酶几何学性质相关的酶解旋酶解旋酶（Helicase）拓扑异构酶拓扑异构酶（Topoisomerases）解旋酶解旋酶（Helicase）至少至少4 4种种helicaseshelicases *reprep helicase helicas

11、e *DNA helicase II *DNA helicase II *DNA helicase III *DNA helicase III *dnaB Protein:dnaB Protein:在在E.coliE.coli DNA DNA复制中解开复制中解开 DNADNA双链双链拓扑异构酶拓扑异构酶（Topoisomerases）拓扑异构酶拓扑异构酶I(topAgene)actonhighlynegativelysupercoiledDNAstabilizesingle-strandedregionsFigure14.16BacterialtypeItopoisomerasesrecogn

12、izepartiallyunwoundsegmentsofDNAandpassonestrandthroughabreakmadeintheother.拓扑异构酶拓扑异构酶IIII TypeIItopoisomerasesgenerallyrelaxbothnegativeandpositivesupercoils.ThereactionrequiresATPFigure14.17TypeIItopoisomerasescanpassaduplexDNAthroughadouble-strandbreakinanotherduplex.拓扑异构酶拓扑异构酶IVIV与子代与子代DNADNA分子的

13、分开有关分子的分开有关参与参与DNA复制的蛋白质复制的蛋白质1 1、参与复制起始的蛋白质因子、参与复制起始的蛋白质因子Originalcomplx:DnaA、DnaB、DnaC、DnaG、HUandSSB Theminimaloriginisdefinedbythedistancebetweentheoutsidemembersofthe13-merand9-merrepeats Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,leadingtotheseparationofDNAstrands.me

14、thylationattheoriginAmembrane-boundinhibitorbindstohemimethylatedDNAattheorigin,andmayfunctionbypreventingthebindingofDnaA.ItisreleasedwhentheDNAisremethylated.SeqAThecomplexatoriCcanbedetectedbyelectronmicroscopy.AntibodiesofdnaAproteinHUThe protein HU is a general DNA-binding protein in E.coli.Its

15、 presence is not absolutely required to initiate replication in vitro,but it stimulates the reaction.HU has the capacity to bend DNA,and is likely to be involved in some general structural capacity.2 2、参与复制延伸的蛋白质因子、参与复制延伸的蛋白质因子(DnaG)(DnaB)2 2、参与参与复制复制终止终止的蛋的蛋白质白质因子因子TusHow do the daughter DNAs becom

16、e disentangled?与真核生物不同，细菌的与真核生物不同，细菌的DNADNA复制复制、染色体重染色体重新折叠以及姊妹染色体的分开新折叠以及姊妹染色体的分开是同时发生的。是同时发生的。细菌中姊妹染色体的分开的机制与真核生物不细菌中姊妹染色体的分开的机制与真核生物不同。细菌染色体的同。细菌染色体的DNADNA分子本身卷入了分开机制。分子本身卷入了分开机制。细菌的多复制叉复制（细菌的多复制叉复制（multiple multiple replicationreplication）与真核生物的复制方式不同。）与真核生物的复制方式不同。*多拷贝的多拷贝的oriCoriC *只有一个终止序列只有一

17、个终止序列Asimplifiedmodelofthebacterialcellcycle.Themodelissimplifiedtoignoremultiforkreplication.SMC类似物类似物A model of a circular chromosome that is undergoing multifork replication in a rod-shaped bacterium.复制起点及终点在细胞中的位置复制起点及终点在细胞中的位置1 1、膜片段中有复制起始区与终止区的富集推断锚定、膜片段中有复制起始区与终止区的富集推断锚定蛋白在定位中的作用。蛋白在定位中的作用。Se

18、qA2 2、位于中间位置、位于中间位置 复制工厂复制工厂 的动力作用的动力作用 多蛋白复合体：多蛋白复合体：polymerase,helicase and accociated polymerase,helicase and accociated proteinsproteins 特殊蛋白质（特殊蛋白质（PolC-GFPPolC-GFP、SeqASeqA)的定位；的定位；H3H3同位素标记；同位素标记；pull DNA template pull DNA template duplicated duplicated release DNA outward during replication

19、release DNA outward during replicationTheextrusion-capturemodelforbacterialchromosomepartitioning.3 3、与染色体组织（、与染色体组织（organizationorganization）、紧结）、紧结(compactioncompaction)和超螺旋和超螺旋(helicasehelicase)有关的蛋白质作用有关的蛋白质作用 SMC SMC MukB MukB to organize the chromosome into a higherto organize the chromosome i

20、nto a higher order structure by constraining supercoils.(order structure by constraining supercoils.(causecause)PartitioningMotorprotein(altered)Chromosomepartitioning(consequence)HU;Hbsu;Terminus-specificchromosomepartitioningevents.Tyrosinesite-specificrecombinasesE.coliCodV,RipXB.subtilisPost-sep

21、tationpartitioningFtsKPBP2RodAPBP3peptidoglycan(肽聚糖肽聚糖)EnvAFigure12.27Failureofcelldivisiongeneratesmultinucleatedfilaments.E.coligenerateanucleatecellswhenchromosomesegregationfails.Cellswithchromosomesstainblue;daughtercellslackingchromosomeshavenobluestain.ThisfieldshowscellsofthemukBmutant;bothn

22、ormalandabnormaldivisionscanbeseen.PhotographkindlyprovidedbySotaHiraga.Minicells:anucleatecellsProblem1.1.哪些哪些DNADNA位点和蛋白质对复制起点的移动及定位起决定性作位点和蛋白质对复制起点的移动及定位起决定性作用？用？2.2.这些位点和蛋白质在指数生长期和进入静止期是否相同？这些位点和蛋白质在指数生长期和进入静止期是否相同？3.3.细胞分裂后，终止序列是怎样定位到细胞的中央部位的？细胞分裂后，终止序列是怎样定位到细胞的中央部位的？4.4.能够进行多复制叉复制的细菌与不能进行多复制叉复制的细能够进行多复制叉复制的细菌与不能进行多复制叉复制的细菌在染色体分开机制上有什么不同？菌在染色体分开机制上有什么不同？5.5.不同物理形状的染色体分开时有差异吗？不同物理形状的染色体分开时有差异吗？20.