Chapter3DNA replication

1、Chapter 03: DNA Replication3.1 The principle of DNA replication3.2 DNA replication model3.3 Enzymes and protein needed in DNA replication3.4 Process of DNA replication3.5 Telomere and Telomerase3.1 The principle of DNA replication3.1.1 semi-conservative replication nDefinition: during replication, t

2、he two parental strands separate and each acts as a template to direct the synthesis of a new complementary daughter strand.nDNA replication pattern: conservative model、semi-conservative model、dispersive modelE.coli ：Radioactive isotope(15N) label CsCl density gradient centrifugationphysical and che

3、mical mutagens DNA damage；During replication or transcription base mismatch；During growth and development modify, deletion, recombinationn Importance: the stability of DNA metabolismn The stability is relative, while the variation is absolute35OK !How ?53533.1.2 Semi-discontinuous replication 35Repl

4、ication forkn1968 Okazaki radioactive label (3H-dTTP) + CsCl density gradient centrifugation label enters newly synthesized DNA in the form of short fragments(1000-2000 bases)nThe ligase temperature-sensitive mutants under the temperature in which the ligase is inactive a large amount of short fragm

5、ents but no lagging strandnConclusion: The lagging strand must be synthesized in the form of Okazaki fragments nOn the leading strand, DNA synthesis can proceed continuously in the 5 to 3 direction as the parental duplex is unwound.nOn the lagging strand, a stretch of single-stranded parental DNA mu

6、st be exposed, and then a segment is synthesized in the reverse direction (relative to fork movement). A series of these fragments are synthesized, then they are joined together to create an intact lagging strand. nOkazaki fragments: the short stretches of 1000-2000 bases in prokaryotes (100-200 bas

7、es in eukaryotes) produced during semi-discontinuous replication, they are later joined into a covalently intact strand. nSemi-discontinuous replication- the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously3.1.3 PrimernA common feature of all DNA pol

8、ymerases is that they cannot initiate synthesis of a DNA chain de novo. nAll DNA polymerases require a 3-OH end to initiate DNA synthesis. nFor DNA replication, a special RNA polymerase called a primase synthesizes an RNA chain(primer) that provides the priming end.E.coli Rif S Rif S + M13E.coliE.co

9、li Rif R + ssDNA virusRF（replicating form）No M13 RFRifampinM13RifampinM13 RFM13 RF M13 RifampinM13expression, packaging, release Its necessary of RNA polymerase to synthesize a RNA in the formation of M13 RF； after the initiation of M13 RF, the inhibition of rifampin is invalidConclusion The first e

10、vidence supporting RNA primer Rifampin is inhibitor of E.coli RNA polymerasenPurpose: keep the high faithfulness nReason: DNA polymerase has proofreading activity, while RNA polymerase dont. After the low-fidelity primer complete its function, it will be replaced by high-fidelity DNA synthesized DNA

11、 polymerase.nResults: improve the accuracy of the DNA replication up to 105 times.n Unidirectionaln Bidirectional(more common)3.1.4 DirectionAutoradiography, Bacillus sp. Gyurasits and Wake3.2 Replication Modeln Replicon：means a sequence from a origin to a terminus.nOrigins tend to be AT-rich to mak

12、e opening easiernAll prokaryotic chromosomes and many phage and viral DNA molecules are circlular and comprise single replicon.nE. coli genome begin bidirectional replication from the unique OriC .There is a single termination site roughly 180o opposite the only origin.nThe long, linear DNA molecule

13、s of eukaryotic chromosomes consist of multiple replicons, each with its own origin.nWhen replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required.3.2.1 formCarinsE.coli DNA3.2.2 Rolling circle formAB3.2.3 D Loop form

14、3.3.1 Enzymes and proteins in pro DNA replication3.3.1.1 DNA polymerases（DNA pols）nThere are at least 5 kinds of DNA pols in E.coli;nReplication- DNA pol IIInDelete primer- DNA pol InRepair- DNA pol II;IV;V3.3 Enzymes and proteins needed in DNA replication表表3-1 大腸桿菌大腸桿菌DNA聚合酶的比較聚合酶的比較n5 3 exonucleas

15、e activity：removing RNA primern35 exonuclease activity: is used to excise bases that have been added to DNA incorrectly proofreading；nKlenow fragment: The larger cleavage product (68 kD) of DNA polby proteolytic treatment (Bacillus subtilis protease), lacking 53 exonuclease activity；DNA polymerase I

16、3 5 exonulease activity: control the fidelity of replication, proofreading ；nNick translation: DNA polymerase I has the ability to start replication in vitro at a nick in DNA. At a point where a phosphodiester bond has been broken in a double-stranded DNA, the enzyme extends the 3-OH end. As the new

17、 segment of DNA is synthesized, it displaces the existing homologous strand in the duplex. nThe major technique for introducing radioactively labeled nucleotides into DNA in vitroPalindromis sequenceRestriction enzymes may give products with 5- or 3- tails or blunt endsSticky endsDNA pol III holoenz

18、ymenThe E.coli DNA polymerase III holoenzyme is a 900 kD complex that contains 10 proteins and organized into four types of subcomplex；nThere are two copies of the catalytic core. Each catalytic core contains a subunit (the DNA polymerase activity), a subunit (35 exonuclease), and a subunit (stimula

19、tes exonuclease)；nThere are two copies of the dimerizing subunit, , which link the two catalytic cores togethernThere are two copies of the clamp, which is responsible for holding catalytic cores on to their template strands. Each clamp consists of a homodimer of subunits that binds around the DNA a

20、nd ensures processivitynThe complex(a group of 5 proteins) is the clamp loader, that places the clamp on DNA the DNA polymerase catalytic core3.3.1.2 HelicasenAn enzyme that separates the strands of DNA, usually using the hydrolysis of ATP to provide the necessary energynIn E.coli ，there are 12 diff

21、erent kinds of helicasesnA helicase is generally multimeric. A common form of helicase is a hexamer.3.3.1.3 single-strand binding protein（SSB） n Binds to the single-stranded DNA, preventing it from reforming the duplex state. n The SSB binds as a monomer, but typically in a cooperative manner ( the

22、binding of additional monomers will improve the stability to the existing complex)n The SSB of E.coli is a tetramer3.1.3.4 DNA ligasenmakes a bond between an adjacent 3 -OH and 5 -phosphate end where there is a nick in one strand of duplex DNAnotices：n the two segments must be complementary to the s

23、ame nucleotide strand；n does not lack of any nucleotiden depends on ATP hydrolysis；35535353HOP DNA ligaseNADATPNMNAMP+PPi+3.3.1.5 TopoisomerasenDNA replication and transcription process are restricted by supercoil nTopoisomerase can regulates DNA supercoil level，by breaking and linking DNA chain to

24、change DNA linking numbernTopoisomerase have endonuclease and ligase activity; can hydrolyze and restore phosphodiester bond；nType cut one chain of the double helix，change 1，dont need energy；Type (gyrase) cut both，change 2，require ATP3.3.1.6 PrimasenThe primase is the product of the dnaG gene. nA si

25、ngle polypeptide of 60 kDa (much smaller than normal RNA polymerase). nDnaG primase associates transiently with the replication complex, and typically synthesizes an 11-12 base primer. 表表3-2 真核生物主要真核生物主要DNA聚合酶的結(jié)構(gòu)及功能聚合酶的結(jié)構(gòu)及功能 3.3.2 enzymes and proteins in eu DNA replication DNADNA聚合酶聚合酶相對(duì)分子質(zhì)量相對(duì)分子質(zhì)量（1

26、0103 3）亞基數(shù)目亞基數(shù)目功能功能3503504 4細(xì)胞核復(fù)制，合成引物細(xì)胞核復(fù)制，合成引物39391 1高忠實(shí)性的修復(fù)高忠實(shí)性的修復(fù)2002002 2線粒體復(fù)制線粒體復(fù)制2502504 4細(xì)胞核復(fù)制，前導(dǎo)鏈合成細(xì)胞核復(fù)制，前導(dǎo)鏈合成3503504 4細(xì)胞核復(fù)制，可能合成后滯鏈細(xì)胞核復(fù)制，可能合成后滯鏈3.3.2.1 DNA pol nDNA pol have primase activity and polymerase activity , but no proofreading activity，it can initiate the synthesis of new strands

27、 (both the leading and lagging strands)nThe pol binds to the initiation complex at the origin and synthesizes a short strand consisting of 10 bases of RNA followed by 20-30 bases of DNA. Then it is replaced by an enzyme that will extend the chain. On the leading strand, this is DNA polymerase - the

28、polymerase switch. nis a highly processive enzyme that continuously synthesizes the leading strand.nIts processivity results from its interaction with two RF-C and PCNA(proliferating cell nuclear antigen)；nThe processivity of DNA pol is maintained by PCNA(processivity unit), which tethers DNA polyme

29、rase to the template.nRF-C is a clamp loader that catalyzes the loading of PCNA on to DNA. 3.3.2.2 DNA pol 3.3.2.3 DNA pol maybe elongate the lagging strand, but it also has been identified with other roles. For example, it can remove primers on Okazaki fragment 3.4.1 prokaryotic DNA replication3.4.

30、1.1 initiationn In E.coli, the replication origin is oriC, 245 bp, whose sequence is very conservative in bacterium.n key sequences: 13bp repeats, 9bp repeats3.4 The process of DNA replicationnThe four 9 bp consensus sequences on the right side of oriC provide the initial binding sites for DnaA. It

31、binds cooperatively to form a central core around which oriC DNA is wrapped. nThen DnaA acts at three AT-rich 13 bp tandem repeats located in the left side of oriC. In the presence of ATP, DnaA melts the DNA strands at each of these sites to form an open complex. n2-4 monomers of DnaA recruit 2 DnaB

32、-DnaC bind to the 13 bp repeats, Each DnaB-DnaC complex consists of 6 DnaC monomers bound to a hexamer of DnaB. nAs DnaB binds, it displaces DnaA from the 13 bp repeats, and extends the region of unwinding by using its helicase activity. nEach DnaB activates a primase(DnaG), initiate the leading str

33、and and the first Okazaki fragment of the lagging strand. nSSB and gyrase are required to support the unwinding reaction. nATP is required at several stages of initiation. The helicase action of DnaB depends on ATP hydrolysis; and the action of topoisomerase requires ATP hydrolysis. ATP is also need

34、ed for the action of primase and to activate DNA pol III.nThere are 11 GATC sequences in the bacterial oriC, Dam methylase can make the adenine to be methylated in N6.nWhen DNA replication is finish, the ori C is half-methylation.nThe half-methylation starting point cant begin another replication, u

35、ntil the oriC is total methylation.nthe lasting time of the half-methylation of the oriC GATC(13min) is much longer than the other parts of the genome GATC (1.5min), except the Dna promoter. It means when the synthesis of the key initiator protein is repressed, the ori C is also inactive.nThe leadin

36、g strand and the lagging strand are replicated by the same DNA polymerase III；none polymerase subunit synthesizes the leading strand continuously, the other cyclically initiates and terminates the Okazaki fragments of the lagging strand within a large single-stranded loop formed by its template stra

37、nd 3.4.1.2 ElongationnThe bacterial chromosome is replicated bidirectionally as a single unit from oriC. nTwo replication forks initiate at oriC and move around the genome (at approximately the same speed) to a meeting point（terminus region）.nE. coli have 5 terminate sites( terA terE), each terminat

38、ion is asymmetric, that is not to keep the opposite side of the replication fork continue replication more than the midpoint.3.4.1.3 terminationnA ter site contains a short (23 bp) sequence that causes termination in vitro. nThe termination sequences function in only one orientation. nThe ter site i

39、s recognized by a protein (called Tus in E.coli, an inhibitor of the DnaB helicase) that recognizes the consensus sequence and prevents the replication fork from proceeding.nAfter the meeting of the two replication forks, replication is stopped. At this time, two circular chromosome become a catenan

40、e. The separation of the catenane needs the topoisomerase IV.3.4.2.1 Originsyeast(S. cerevisiae)nA high-frequency transforming fragment possesses a sequence that confers the ability to replicate efficiently in yeast. nThis segment is called an ARS(autonomously replication sequence). ARS elements are

41、 derived from replication origins.3.4.2 key notes of Eukaryotic DNA replicationnOrigins in yeast are short AT-rich sequences that have an essential 11 bp sequence.nThe ORC(origin recognition complex) is a complex of 6 proteins with a mass of 400 kDa that binds to an ARS.nIn some cases, many small re

42、plication bubbles are found in one region, which means that eukaryotes maybe have alternative multiple starts to replication.nAt one location where multiple bubbles are found, there is a primary origin that is used predominantly when the nucleotide supply is high. nBut when the nucleotide supply is

43、limiting, many secondary origins are also used, giving rise to a pattern of multiple bubbles.nLicense factors (MCM2MCM7) are necessary for replication initiation. nBinds to chromosomal material before replication, and degraded rapidly after replication. nOnly during the next cell division, the licen

44、se factors will enter the nucleus again.DNA replication in eukaryotic cells- yeast repliconARS: Origins of replicationORC: Origin recognition complexMCM: Licensing factor3.4.2.2 Elongation and termination SV40nDifferent from prokaryotic DNA replication, Eukaryotic DNA replication has no distinct ter

45、mini.nReplication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. nReplication separates the strands of DNA and therefore must inevitably disrupt the structure of the nucleosome. nReproduction of chromatin does not involve any protracted period during w

46、hich the DNA is free of histones. Once DNA has been replicated, nucleosomes are quickly generated on both the duplicates. 3.4.2.3 the histone replicationExperiment:nCells are grown in the presence of heavy amino acids(15N) before replication. Then replication is allowed to occur in the presence of l

47、ight amino acids(14N) nAt the point the histone octamers are crosslinked and centrifuged on a density gradientnThe octamers have an intermediate densityExplain: the old histones have been released and then reassembled with newly synthesized histones.3-OHcircular DNA replication linear DNA replicatio

48、n But 35533-OH ? 3-OH ? The ends of linear DNA are a problem for replication3.5.1 structure of the telomerenTelomere is the natural end of chromosome.nEach telomere consists of a long series of short, tandemly repeated sequences. There may be 100-1000 repeats, depending on the organism. nOne strand has the general sequence Cx(A/T)y, where x1 and y = 1-4. The other strand, Gx(T/A)y, has a single protruding end (3) that