參與原核生物DNA復(fù)制的

1、參與原核生物參與原核生物DNADNA復(fù)制的復(fù)制的酶類和蛋白質(zhì)酶類和蛋白質(zhì)Enzymes and Proteins Involved in DNA Enzymes and Proteins Involved in DNA Replication in ProkaryotesReplication in Prokaryotes 高方遠(yuǎn)高方遠(yuǎn) 馬欣榮馬欣榮 康海岐康海岐DNA replication(bacteria)InitiationElongationTerminationDaughter DNA partition* the origin of replication is defined

2、* replication bubble. * Replication fork* Unidirectional or bidirectional.OriginsFigure 13.9 The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously. Elongation(semidiscontinuous)termination參與參與DNA復(fù)制的酶類復(fù)制的酶類1 1、DNADNA聚合酶聚合酶2 2、DNADNA引發(fā)酶（引發(fā)酶（DNA primaseD

3、NA primase）3 3、DNADNA連接酶連接酶4 4、與、與DNADNA幾何學(xué)性質(zhì)相關(guān)的酶幾何學(xué)性質(zhì)相關(guān)的酶 I polA major repair enzyme II polB minor repair enzyme III polC replicase IV dinB SOS repair V umuC、D SOS repairDNA polymerases in E.coliDNA Polymerase I DNA polymerase3 5 exonuclease5 3 exonucleaseFigure 13.8 The catalytic domain of a DNA p

4、olymerase has a DNA-binding cleft created by three subdomains. The 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

5、 fingers and thumb. Photograph kindly provided by Charles Richardson 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 m

6、ass function subassemblies (KDa) 129.9 DNA polymerase 27.5 3 5 exonuclease 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 II

7、I Beta-subunitFigure 13.18 DNA polymerase III holoenzyme assembles 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

8、 is represented by the red rectangle. The three major steps in TLR 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). SS

9、B is suggested to help in displacing RecA from DNA both at the initiation and lesion bypass steps. E. coli DNA polymerase IV （ dinB gene ） * dinB dinB 基因的表達(dá)需要基因的表達(dá)需要 DNADNA損傷誘導(dǎo)損傷誘導(dǎo) * * 與與UmuCUmuC、 UmuDUmuD同屬同屬Y Y 家族家族DNADNA聚合酶聚合酶 * E. coli DNA polymerase IV無校正功能，易于延長一些凸無校正功能，易于延長一些凸出的引物或模板結(jié)構(gòu)。出的引物或模板

10、結(jié)構(gòu)。2 2、DNADNA引發(fā)酶（引發(fā)酶（DNA primaseDNA primase） Use host RNA polymerase 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 syn

11、thesis of the primer. Adenovirus terminal protein binds to the 5 end 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 Poly

12、merase I與與DNADNA幾何學(xué)性質(zhì)相關(guān)的酶幾何學(xué)性質(zhì)相關(guān)的酶解旋酶解旋酶（Helicase）拓?fù)洚悩?gòu)酶拓?fù)洚悩?gòu)酶（Topoisomerases）解旋酶解旋酶（Helicase）至少至少4 4種種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復(fù)制中解開復(fù)制中解開 DNADNA雙鏈雙鏈拓?fù)?/p>

13、異構(gòu)酶拓?fù)洚悩?gòu)酶（Topoisomerases）拓?fù)洚悩?gòu)酶拓?fù)洚悩?gòu)酶I(topA gene) act on highly negatively 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?fù)洚悩?gòu)酶拓?fù)洚悩?gòu)酶IIII Type II topoisomerase

14、s generally relax both negative and positive supercoils. The reaction requires ATPFigure 14.17 Type II topoisomerases can pass a duplex DNA through a double-strand break in another duplex. 拓?fù)洚悩?gòu)酶拓?fù)洚悩?gòu)酶IVIV與子代與子代DNADNA分子的分開有關(guān)分子的分開有關(guān)參與參與DNA復(fù)制的蛋白質(zhì)復(fù)制的蛋白質(zhì)1 1、參與復(fù)制起始的蛋白質(zhì)因子、參與復(fù)制起始的蛋白質(zhì)因子Original complx: DnaA、D

15、naB、DnaC、 DnaG、 HUand SSB The minimal origin is defined by the distance 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 i

16、nhibitor binds to hemimethylated DNA at the origin, and may function 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

17、. Its 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、參與復(fù)制延伸的蛋白質(zhì)因子、參與復(fù)制延伸的蛋白質(zhì)因子(Dna G)(DnaB)2 2、參與參與復(fù)制復(fù)制終止終止的蛋的蛋白質(zhì)白質(zhì)因子因子TusHow do the daughter

18、DNAs become disentangled? 與真核生物不同，細(xì)菌的與真核生物不同，細(xì)菌的DNADNA復(fù)制復(fù)制、染色體重染色體重新折疊以及姊妹染色體的分開新折疊以及姊妹染色體的分開是同時(shí)發(fā)生的。是同時(shí)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)菌染色體的同。細(xì)菌染色體的DNADNA分子本身卷入了分開機(jī)制。分子本身卷入了分開機(jī)制。 細(xì)菌的多復(fù)制叉復(fù)制（細(xì)菌的多復(fù)制叉復(fù)制（multiple replicationmultiple replication）與真核生物的復(fù)制方式不同。與真核生物的復(fù)制方式不同。 * * 多拷貝的多拷貝的oriCo

19、riC * * 只有一個(gè)終止序列只有一個(gè)終止序列A simplified model of the bacterial cell cycle.The model is simplified to ignore multifork replication.SMC類似物類似物 A model of a circular chromosome that is undergoing multifork replication in a rod-shaped bacterium. 復(fù)制起點(diǎn)及終點(diǎn)在細(xì)胞中的位置復(fù)制起點(diǎn)及終點(diǎn)在細(xì)胞中的位置1 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、膜片段中有復(fù)制起

20、始區(qū)與終止區(qū)的富集推斷錨定蛋白在定位中的作用。蛋白在定位中的作用。SeqA2 2、位于中間位置、位于中間位置 復(fù)制工廠復(fù)制工廠 的動(dòng)力作用的動(dòng)力作用 多蛋白復(fù)合體：多蛋白復(fù)合體：polymerase, helicase and accociated polymerase, helicase and accociated proteinsproteins 特殊蛋白質(zhì)（特殊蛋白質(zhì)（PolC-GFPPolC-GFP、SeqASeqA) ) 的定位；的定位；H3H3同位素標(biāo)記；同位素標(biāo)記； pull DNA templatepull DNA template duplicated duplicated

21、 release DNA outward during replication release DNA outward during replicationThe extrusion-capture model for bacterial chromosome partitioning.3 3、與染色體組織（、與染色體組織（organizationorganization）、緊結(jié)）、緊結(jié)( (compactioncompaction) )和超螺旋和超螺旋( (helicasehelicase) )有關(guān)的蛋白質(zhì)作用有關(guān)的蛋白質(zhì)作用 SMCSMC MukB MukB to organize the

22、 chromosome into a higherto organize the chromosome into a higher order 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-se