MCB 250 Lecture 11 DNA Replication in BacteriaSlide 2Slide 3Slide 4Are Eukaryotic Chromosomes Supercoiled?Wrapping of DNA Around Histones Introduces Negative SupercoilsSlide 7Slide 8Scale of the ProblemDNA Replication is SemiconservativeSlide 11Replication proceeds 5’ to 3’The substrates for DNA replication are dNTPsChemistry of DNA ReplicationSlide 15DNA Replication is Semi-continuousSlide 17Slide 18Proteins Required for ReplicationHelicase - DnaBSlide 21Slide 22Slide 23PrimaseWhy Use RNA as a PrimerE. coli DNA Polymerase IIIPol III CoreThe Polymerization ReactionSlide 29Clicker QuestionDNA Replication is Highly AccurateSlide 32Slide 33Slide 34Subunit: The ClampSlide 36Importance of Being Processiveg Subunit: The Clamp LoaderSlide 39DNA PolIII HoloenzymeProtein Interactions at the Replication ForkSlide 42Slide 43RNAse HE. coli Polymerase I (Pol I)Slide 46DNA LigaseDNA LigaseMCB 250 Lecture 11 DNA Replication in BacteriaThe Level of DNA Compaction and Position of Histones is Highly Regulated•In states more compact than the 10 nm fiber, DNA is not accessible for transcription, replication, or repair.•Using the light microscope, cytologists have observed for many years that part of the chromatin in the nucleus (heterochromatin) is condensed and stains densely with many dyes. Some of the chromatin (euchromatin) stains weakly with dyes and appears to have an open structure. Little or no transcriptionActive transcriptionLow level transcriptionHeterochromatin EuchromatinGRADED EFFECTThe Level of DNA Compaction and Position of Histones is Highly Regulated•We now understand that the DNA heterochromatic regions is poorly transcribed whereas genes that are highly expressed (frequently transcribed) are in euchromatic regions. •So chromatin structure can determine which genes are turned off and which are turned on.•Even regions of DNA that are not transcribed must be made accessible for DNA replication and repair.•So, how can chromatin structure be altered to allow access to the DNA?Regulation of Chromatin Structure (The simple version)Fig 8-39•The overall accessibility of the DNA can be modified•Individual histones can be moved to free up a binding site for a regulatory protein•Much of this is controlled by proteins that specifically modify the histone tails, giving a “histone code”•We’ll talk about it laterAre Eukaryotic Chromosomes Supercoiled?•Eukaryotic chromosomes are linear – not covalently closed circles.•But DNA strands are very long and therefore are topologically constrained.•Are the ends attached to anything?–Yes – maybe the nuclear matrix.•But wrapping around the histones is equivalent to negative supercoiling.•Eukaryotes have Type I and II topoisomerases.Wrapping of DNA Around Histones Introduces Negative SupercoilsDNA Replication in BacteriaOverview of DNA ReplicationE. coli ChromosomeSize of Intact CellDNA is 1 mmCell is 1 mmScale of the Problem •E. coli chromosome is 4.5 x 106 basepairs and is 1 mm long•If the DNA was a 2-stranded kite string 1 mm wide, then it would be ½ mile long and the cell would be ¾ of a meter in diameter–Your assignment: separate the two strands, wrap a new strand around each and end up with two new pieces and NO knots. You have 40 minutes with an average error rate (incorporation of a wrong base, i.e. a mutation) of once per 109 bases. That’s 1 mistake ~every 1000 chromosomes.DNA Replication is SemiconservativeThe products of replication are duplexes with one old strand (blue) and one new one (red).Origin ofReplicationResolutionChromosomal Replication is BidirectionalTwo ReplicationForksAll DNA polymerases require a primer and a template. The primer grows, the template is copied. Synthesis is in the 5’ to 3’ direction. This means that the 3’ end grows.Replication proceeds 5’ to 3’The building blocks for DNA replication are the four deoxynucleoside triphosphates (dNTPs where N = A,T,G, or C). The substrates for DNA replication are dNTPsChemistry of DNA ReplicationThe 3’-hydroxyl of the growing chain carries out a nucleophilic attack on the -phosphate of the incoming NTP forming a new phosphodiester bond.The other product is pyrophosphate (PP) which is rapidly converted to phosphate by pyrophosphatase. Hydrolysis of the PP product drives the reaction in the direction of polymerization.Fig. 9-2DNA Replication is Semi-continuousDirection of forkmovementParental DNA duplexDaughter duplexLeading strandShort RNA primerOkazaki fragmentLagging strandPoint of joining5’3’3’5’5’DNA replicationAfter Passage, Pol I Removes RNA primer and Completes DNA SynthesisDNA Ligase Seals NicksOkasaki FragmentsReplisome (2xPol III) Synthesizes both Leading and Lagging Strands at Replication Fork5’3’5’3’POHNick5’3’5’3’The ReplisomeLeading StrandDNA PolymeraseLagging StrandDNA PolymeraseSliding Clampg-Complext-ProteinDNA HelicaseSSB BoundTo DNA5’3’3’5’3’5’3’5’3’OHPrimaseNote that the leading strand polymerase and the lagging strand polymerase are connected by interactions with the t-protein of the clamp loader. 5’Proteins Required for Replication•Helicase (DnaB)–Melts parental DNA, interacts with PolIII and Primase•Primase (DnaG)–Synthesizes RNA primers•DNA Polymerase III (Pol III)–Synthesizes DNA. Requires a primer.•Single Strand Binding Protein (SSB)–Binds single stranded DNA template cooperatively, prevents reannealing and hairpins•RNAse H–Removes RNA primers•DNA Polymerase I (Pol I)–Removes RNA primers and replaces RNA with DNA •DNA ligase–Seals nicks between Okazaki fragments•Topoisomerase–Relaxes DNA in front of the replication forkHelicase - DnaB•Hexamer – 6 identical subunits•Wraps completely around the lagging strand•Requires DnaC to load•Travels 5’ to 3’•Uses ATP to unwind the helixElectron MicroscopeReconstructionHelicase is Required to Unwind the DNA DuplexThe helicase travels 5’3’ on the lagging strand template.Fig 9-14Single Stranded Binding Protein (Ssb) Prevents Re-annealing of the HelixBinding of SSB is Highly Cooperative - Binding of one SSB makes it likely that another will bind next to itFig 9-16Primase•Recruited by Helicase•Starts preferentially at 5’-CTG-3’ and lays down a 10-12 nucleotide primer•Then dissociates – not processive•Must act every 1- 2 kb or so on lagging strandActive site cleftWhy Use RNA as a Primer•We don’t really know•Leftover