DNA Replication  The basic rules for DNA replication  DNA synthesis at the replication fork  Termination of replication  Other modes of DNA replication

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  • DNA Replication The basic rules for DNA replication DNA synthesis at the replication fork Termination of replication Other modes of DNA replication DNA Polymerases Initiation of replication Regulation of re-initiation

  • Initiation of replication Common features of replication origins Common events of initiation Priming

  • (proposed by F. Jacob, S. Brenner and J. Cuzin, 1963)All the DNA replicated from a particular origin as a replicon. Binding of the initiator to the replicator stimulates initiation of replication. The replicon model of replication initiation:

  • ReplicatorReplicator: the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of DNA replication (*the origin of replication is part of replicator)InitiatorInitiator: the DNA-binding protein that specifically recognizes a DNA element in the replicator and activates the initiation of replication

  • Initiator binding siteEasily melted regionReplicatorInitiatorDNA bindingDNA unwindingProtein recruitmentPriming and DNA synthesisThe functions of initiator(Origin)

  • DNA bindingDNA unwindingProtein recruitmentPriming and DNA synthesisoriCDnaADnaB/DnaCE. coli

  • oriC : the origin of replication in E. coliEasily melted(AT rich)Initiator (DnaA) binding siteFigure 14.26

  • Consensus sequenceGATCTNTTNTTTTCTAGANAANAAAAConsensus sequenceTTATNCANAAATANGTNTL, M, R repeats (13 bp)1~4 repeats (9 bp) oriC:

  • The replicator (origin) of S. cerevisiae:Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17 Origin recognition complex (ORC)(Initiation complex)ACS: ARS consensus sequenceARS (Autonomously replicating sequence)

  • Mutation in ARSMutations in B elements reduce origin function.Mutations in core consensus abolish origin function.Figure 13.20

  • DNA bindingDNA unwindingProtein recruitmentPriming and DNA synthesisoriCDnaADnaB/DnaCE. coli

  • Figure 14.27Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-26.DNA helicase(DnaB)DNA helicaseLoader (DnaB)DnaA.ATP

  • DnaAATPHU + ATP(histone like protein)

  • DNA helicase(DnaB)DNA helicaseLoader (DnaB)

  • Figure 14.10

  • Two types of function are needed to convert dsDNA to the single-stranded state: 2. Single-strand binding proteins bind to the ssDNA, preventing it from reforming the duplex state Helicases separate the strands of DNA, usually using the hydrolysis of ATP to provide the necessay energy

  • Single-strand binding proteinsDNA helicase(DnaB)DNA helicaseLoader (DnaB)Primase

  • For DNA replication, a primase is required to catalyze the synthesis of RNA primer.Primase in E. coli: An RNA polymerase Encoded by the dnaG gene Synthesizing short stretches of RNA

  • Figure 14.14

  • Protein required to initiate replication at the E. coli origin: Summary

    Protein FunctionDnaA protein Recognizes origin sequence; open duplex at specific sites in originDnaB protein (helicase) Unwinds DNADnaC protein Required for DnaB binding at originHU DNA bending protein; stimulates initiationPrimase (DnaG protein) Synthesizes RNA primersSingle-strand DNA-binding protein (SSB) Binds single-strand DNADNA gyrase(DNA topoisomerase) Relieves torsional strain generated by DNA unwindingDam methylase Methylates 5-GATC-3 sequences at oriC

  • DNA Replication The basic rules for DNA replication DNA synthesis at the replication fork Termination of replication Other modes of DNA replication DNA Polymerases Initiation of replication Regulation of re-initiation

  • DNA synthesis at the replication fork Proteins at the replication forks Coordinating synthesis of the lagging and leading strands in eukaryotic cells in E. coli

  • DNA replication is semidiscontinuous.Figure 14.91000-2000 nt in prokaryotes100-400 nt in eukaryotesOkazaki fragments

  • *SSB: Single-strand binding proteinsProteins required at the replication forks:Primer removal enzyme DNA ligase

  • Different replicase units are required to synthesize the leading and lagging strands.In E. coli both units contain the same catalytic subunit of DNA Pol III. In other organisms, different catalytic subunits may be required for each strand.

  • The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits. Each polymerase catalytic subunit is held on DNA by a sliding clamp. Figure 14.19

  • The polymerase that synthesizes the lagging strand dissociates at the end of Okazaki fragment and then reassociates with a primer in the single- stranded template loop to synthesize the next fragment. The polymerase that synthesizes the leading strand moves continuously. Figure 14.19

  • In E. coli:DnaBDnaGDNA Pol IIIholoenzyme

  • E. coli DNA Polymerase III holoenzymeBased on Figure 14.17

  • eeqqaattCore enzymeproofreadingpolymerizationpolymerizationCore enzyme dimerization

  • Clamp loaderATPATPADPATPSliding clampATPhydrolysisPibb

  • Core enzyme: 10 ~ 15 Holoenzyme: >500000b converts Pol III from a distributive enzyme to a highly processive enzyme.ProcessivityFigure 14.18

  • ygddeeqqaattct subunits maintain dimeric structure of Pol III and interact with DnaBDnaB(helicase)Lagging strand synthesisLeading strand synthesis

  • Each catalytic core of polymerase III synthesizes a daughter strand. DnaB (helicase) is responsible for forward movement at the replication fork.Figure 14.20

  • What happens to the loop when the Okazaki fragment is completed? Figure 14.21

  • Initiation of Okazaki fragment Termination of Okazaki fragmentDissociation of core and b clampReassociation of b clamp1234Figure 14.205. Reassociation of core

  • Each Okazaki fragment is synthesized as a discrete unit.Primase synthesizes RNA primer.DNA Pol III extends primer into Okazaki fragment.Next Okazaki fragment is synthesized.Leading strandLagging strand

  • Okazaki fragments are linked together. DNA Pol I uses nick translation to replace RNA primer with DNA.Ligase seals the nick.Figure 14.225335RNA primer

  • 53 Exonuclease activity of DNA Pol I: Figure 14.5

  • +Adenylylation of DNA ligase1Mechanism of the DNA ligase reaction+ NMN (or PPi)

  • LigaseNH2+POO-ORiboseAdenineLigaseActivation of 5 phosphate in nick23Figure 14.23*

  • POO-OOPOO-ORiboseAdenineThe 3-hydroxyl group attacks the phosphate and displaces AMP, producing a phosphodiester bond. 33

  • Pola/primaseEukaryotic cell(RFC)PCNA Pold Pold/e

  • Eukaryotes have many DNA polymerases.Figure 14.24

  • Eukaryotic DNA polymerases for replication in nucleus:

    DNApolymerasePrimaseactivity ProcessivityProof-readingFunctiona+moderate-Primer synthesisd-High+Leading/lagging? strand synthesise-High+laggingstrand synthesis

  • DNA polymerase a (Pola/primase):2 subunits: Pol a2 subunits: primase DNA synthesisRNA synthesis35RNADNA (iDNA)53OH~ 10 bp20-30 bp

  • DNA polymerase switching during eukaryotic DNA replication:DNA Pol a/ primaseRNA primer synthesis by primase DNA synthesis by Pol aPaRNAiDNAWatson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-16.

  • SlidingclampPola/primaseDNA Pol e (or d)iDNA*

  • R-FC binds to the 3 end of iDNA and displaces pol a/primasePCNA binds pol d or eRF-C: Clamp loaderPCNA: Sliding clamp*R-FC attracts PCNA

  • PCNA Proliferating cell nuclear antigenVoet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-1(trimer)

  • Proteins required at the replication forks:SummaryDNA topoisomerases are also required!Figure 14.25

  • There are two ways to think of the relative motion of the DNA and replication machinery: The replication machinery moves along the DNA. (similar to a train moving along its track)2. The DNA moves while the replication machinery is static. (similar to film moving into a movie projector)

  • The two replisomes of E. coli are linked together and tethered to one point on the bacterial inner membrane. Helicases(double-hexamers)Pol III holoenzymePol III holoenzyme Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a

  • OriginTerminatorChromosomeReplisomesReplication beginsOrigins separateCell elongates as replication continuesChromosomes separateCells divide Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a

    Binding of DnaA, plus the basic proteins, bends the DNA rather sharply and creates negative super-helical tension. In turn this tension causes DNA unwinding in the 13 bp regions.

    The timing of replication initiation is affected by DNA methylation and interactions with the bacterial plasma membrane. The oriC region of E.coli is highly enriched in GATC sequences, containing 11 of them in its 245 bp.( See below for regulation of re-initiation).