最新参与原核生物DNA复制的酶类和蛋白质EnzymesandProteinsppt课件.ppt

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1、参与原核生物参与原核生物DNA复制的酶类复制的酶类和蛋白质和蛋白质EnzymesandProteinsDNA replication(bacteria)InitiationElongationTerminationDaughter DNA partitionDNA Polymerase I DNA polymerase3 5 exonuclease5 3 exonucleaseFigure 13.8 The catalytic domain of a DNA polymerase has a DNA-binding cleft created by three subdomains. The

2、active site is in the palm. Proofreading is provided by a separate active site in an exonuclease domain. Figure 13.7 Crystal structure of phage T7 DNA polymerase has a right hand structure. DNA lies across the palm and is held by the fingers and thumb. Photograph kindly provided by Charles Richardso

3、n and Tom Ellenberger. Figure 13.5 Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material. DNA Polymerase ISubunit composition of E.coli DNA polymerase III holoenzyme subunit molecular mass function subassemblies (KDa) 129.9 DNA polymerase 27.5 3 5 exon

4、uclease core 8.6 stimulates exonuclease 71.1 dimerizes core Pol III binds complex 47.5 binds ATP 38.7 binds to Pol III 36.9 binds to and complex 16.6 binds to SSB DNA-dependent 15.2 binds to and ATPase 40.6 sliding clampE.coli Pol III Beta-subunitFigure 13.18 DNA polymerase III holoenzyme assembles

5、in stages, generating an enzyme complex that synthesizes the DNA of both new strands. Fig. 1. Model of SOS translesion replication by DNA polymerase V. The two DNA strands are shown as green lines, and the replication-blocking lesion is represented by the red rectangle. The three major steps in TLR

6、are pre-initiation (2), in which the RecA nucleoprotein filaments assembles; initiation (3 and 4), which involves binding of pol V to the primer-template and loading of the subunit clamp; and lesion bypass by pol V holoenzyme (5). SSB is suggested to help in displacing RecA from DNA both at the init

7、iation and lesion bypass steps. E. coli DNA polymerase IV （ dinB gene ） * dinB dinB 基因的表达需要基因的表达需要 DNADNA损伤诱导损伤诱导 * * 与与UmuCUmuC、 UmuDUmuD同属同属Y Y 家族家族DNADNA聚合酶聚合酶 * E. coli DNA polymerase IV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） Use host RNA polymeras

8、e as primase (M13) primosome primase (dnaG protein) (E.coli) other proteins X174: only primase, without the other proteins Initiation requires several enzymatic activities, including helicases, single-strand binding proteins, and synthesis of the primer. Adenovirus terminal protein binds to the 5 en

9、d of DNA and provides a C-OH end to prime synthesis of a new DNA strand. A primer terminus is generated within duplex DNA.Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material. DNA Polymerase I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（Topois

10、omerases）解旋酶解旋酶（Helicase）至少至少4 4种种helicaseshelicases * * reprep helicase helicase * * 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(topA gene) act on highly negati

11、vely supercoiled DNA stabilize single-stranded regionsFigure 14.16 Bacterial type I topoisomerases recognize partially unwound segments of DNA and pass one strand through a break made in the other.拓扑异构酶拓扑异构酶IIII Type II topoisomerases generally relax both negative and positive supercoils. The reacti

12、on requires ATPFigure 14.17 Type II topoisomerases can pass a duplex DNA through a double-strand break in another duplex. 拓扑异构酶拓扑异构酶IVIV与子代与子代DNADNA分子的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Original complx: DnaA、DnaB、DnaC、 DnaG、 HUand SSB The minimal origin is defined by the dist

13、ance between the outside members of the 13-mer and 9-mer repeats Prepriming involves formation of a complex by sequential association of proteins, leading to the separation of DNA strands. methylation at the origin A membrane-bound inhibitor binds to hemimethylated DNA at the origin, and may functio

14、n by preventing the binding of DnaA. It is released when the DNA is remethylated. SeqAThe complex at oriC can be detected by electron microscopy. Antibodies of dnaAprotein HUThe protein HU is a general DNA-binding protein in E. coli . Its presence is not absolutely required to initiate replication i

15、n 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(Dna G)(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 become 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

16、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 replicationmultiple replication）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个终止序列A simplified model of the bacterial cell cy

17、cle.The model is simplified to ignore multifork replication.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。SeqA2 2、位于中间位置、位于中间位置 复制工厂复制工厂 的动力

18、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 templatepull DNA template duplicated duplicated release DNA outward during replication release DNA outward during

19、replicationThe extrusion-capture model for bacterial chromosome partitioning.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 SMCSMC MukB MukB to organize the chromosome into a higherto organize the chromosome into a higher o

20、rder structure by constraining supercoils.( order structure by constraining supercoils.(causecause) ) Partitioning Motor protein (altered)Chromosome partitioning(consequence)HU; Hbsu ;Terminus-specific chromosome partitioning events. Tyrosine site-specific recombinasesE.coliCodV, RipXB.subtilisPost-

21、septationpartitioningFtsKPBP2 RodAPBP 3 peptidoglycan(肽聚糖肽聚糖)EnvAFigure 12.27 Failure of cell division generates multinucleated filaments. E. coli generate anucleate cells when chromosome segregation fails. Cells with chromosomes stain blue; daughter cells lacking chromosomes have no blue stain. Thi

22、s field shows cells of the mukB mutant; both normal and abnormal divisions can be seen. Photograph kindly provided by Sota Hiraga. Minicells: anucleate cellsProblem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？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？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？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？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？52结束语结束语