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Chapter 10 Replication of DNA
© John Wiley & Sons, Inc.
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Chapter Outline Basic Features of DNA Replication In Vivo
DNA Replication in Prokaryotes Unique Aspects of Eukaryotic DNA Replication © John Wiley & Sons, Inc.
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Basic Features of DNA Replication In Vivo
DNA replication occurs semiconservatively, is initiated at unique origins, and usually proceeds bidirectionally from each origin of replication. Synthesis of DNA (RNA,proteins): 1-initiation, 2-extension/elongation, 3-termiantion. DNA polymerase (protein-enzyme)-essential for conservation of any species 3,000/30,000 nucleotides per minutes One mistake per billion of nucleotides © John Wiley & Sons, Inc.
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DNA Replication is Semiconservative
MODEL Each strand serves as a template Complementary base pairing determines the sequence of the new strand Each strand of the parental helix is conserved Semiconservative=half conserve © John Wiley & Sons, Inc.
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Possible Models of DNA Replication
© John Wiley & Sons, Inc.
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CsCl Equilibrium Density-Gradient Centrifugation
Bacteria growing with 15N and 14N (normal) Density: GsCl ~1.7 g/cm3 DNA ~1.710 g/cm3 with 14N ~1.724 g/cm3 with 15N Bacteria with 15N-parental (several periods of time) Bacteria with 14N-daugther Centrifugation: process involving the centrifugal force for the sedimentation of particles and/or molecules [revolutions per minute (RPM)] gravitational force to cause precipitation/sedimentation © John Wiley & Sons, Inc.
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The Meselson-Stahl Experiment: DNA Replication in E
The Meselson-Stahl Experiment: DNA Replication in E. coli is Semiconservative Bacteria growing with 15N for several generations Change medium and add 14N --one generation --two generations --three generations © John Wiley & Sons, Inc.
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Semiconservative Replication in Eukaryotes
Autoradiography: A technique using X- ray film to visualize molecules or fragments of molecules that have been radioactively labeled 1H-Thymidine (normal) 3H-Thymidine Autoradiography 3H=tritium © John Wiley & Sons, Inc.
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C-metaphase: Colchicine-metaphase:
3H-Thymidine 1H-Thymidine C-metaphase: Colchicine-metaphase: Colchincine: is a toxic natural product and secondary metabolite and it inhibits microtubule polymerization by binding to tubulin. © John Wiley & Sons, Inc.
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The Origin of Replication in E. coli
Replicon: is a sequence of DNA at which DNA replication is initiated on a chromosome, plasmid or virus. -OriC (245 bp) -AT-rich region (replication bubble) -13-mer and 9-mer tandem N: any nucleotide Eukaryotic: ARS (Autonomously Replicating sequences) AT-rich region 11bp Mer=repeating unit=parts © John Wiley & Sons, Inc.
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Bidirectional Replication of the Circular E. coli Chromosome
-Circular DNA (double strand DNA) --Unwind (access and single strand DNA) --Simultaneous semiconservative replication --Swivel (point of break) Topoisomerases --Y-shape structure=replication fork Topoisomerases: are enzymes that regulate the overwinding or underwinding of DNA. © John Wiley & Sons, Inc.
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Visualization of Replication in E. coli
3H-Thymidine Autoradiography © John Wiley & Sons, Inc.
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Visualization of Replication in E. coli
3H-Thymidine Autoradiography © John Wiley & Sons, Inc. 20
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Bidirectional Replication: The Phage Chromosome
-Small bacterial virus Single stranded DNA (12 bp) Cohesive/sticky and complementary ends DNA ligase (replication, repair and recombination) Linear Circular replication © John Wiley & Sons, Inc.
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Replication is Bidirectional
1-AT- and CG -rich regions Native Denature 2-Bubbles 3-DNA polymerase access 100°C pH~11 ~10 min © John Wiley & Sons, Inc.
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Replication is Bidirectional
Both branch points ( Y shape) are replication forks Replication fork: junction where the double-stranded DNA splits apart (or unzipped) into 2 single strands. © John Wiley & Sons, Inc.
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Bidirectional Replication of Phage T7
--eye structure replication forks © John Wiley & Sons, Inc.
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DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix unwind and separate, each serves as a template for the synthesis of a new complementary strand. The hydrogen-bonding potentials of the bases in the template strands specify complementary base sequences in the nascent DNA strands. Replication is initiated at unique origins and usually proceeds bidirectionally from each origin. © John Wiley & Sons, Inc.
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DNA Replication in Prokaryotes
DNA replication is a complex process, requiring the concerted action of a large number of proteins © John Wiley & Sons, Inc.
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DNA Polymerases and DNA Synthesis In Vitro
Much of what we know about DNA synthesis was deduced from in vitro studies. DNA Polymerase I Single polypeptides 5’ to 3’ Triphosphate [dATP] MgCl2 Free 3’OH group of the DNA strands © John Wiley & Sons, Inc.
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Continuous vs discontinuous--leading and lagging strands
Replicating fork Bacteriophage T4 © John Wiley & Sons, Inc.
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Small fragments to big fragments
1,000 to 2,000 bp Okazaki fragments 10,000 to 200,000 bp Small fragments to big fragments © John Wiley & Sons, Inc.
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Continuous vs discontinuous--leading and lagging strands
Replicating fork © John Wiley & Sons, Inc. 30
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© John Wiley & Sons, Inc.
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Prepriming at oriC in E. coli
--Replication bubbles Self aggregation DNA helicase: it separates two annealed nucleic acid Strands. Why? © John Wiley & Sons, Inc.
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RNA Primers are Used to Initiate DNA Synthesis
DNA primase: short RNA primer RNA/DNA hybrid (unstable ?) Perfect conditions for DNA polymerases to work (free 3’OH) © John Wiley & Sons, Inc.
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DNA Polymerase I: 5'3' Polymerase Activity
© John Wiley & Sons, Inc.
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DNA Polymerase I: 5'3' Exonuclease Activity
© John Wiley & Sons, Inc.
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DNA Polymerase I: 3'5' Exonuclease Activity
© John Wiley & Sons, Inc.
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© John Wiley & Sons, Inc.
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DNA Helicase Unwinds the Parental Double Helix
One of the most important event during DNA replication Why? © John Wiley & Sons, Inc.
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Single-Strand DNA Binding (SSB) Protein
Access to DNA polymerase © John Wiley & Sons, Inc.
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Supercoiling of Unwound DNA
DNA Topoisomerases I: produce single transient breaks of DNA and remove supercoiling It blocks DNA replication DNA Topoisomerases II: produce double transient breaks of DNA and negative supercoiling (DNA gyrase) © John Wiley & Sons, Inc.
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DNA Topoisomerase I Produces Single-Strand Breaks in DNA
© John Wiley & Sons, Inc.
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Requirements of DNA Polymerases
Primer DNA with free 3'-OH Template DNA to specify the sequence of the new strand Substrates: dNTPs Mg2+ Reaction: nucleophilic attack © John Wiley & Sons, Inc.
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DNA Polymerase III is the True DNA Replicase of E. coli
--a 900 KDa multimeric protein --Dimers --Holoenzymes --High fidelity (error ~1 in a 1 x 1012) © John Wiley & Sons, Inc.
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Proofreading mechanism
Subunits----Prokaryotes Subunits----Eukaryotes © John Wiley & Sons, Inc.
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The Replication Apparatus in E. coli
Primosome: Initiation of Okazaki fragment during lagging strand DNA primase and DNA helicase DnaB and C proteins Require ATP DNA helicase:unwinds DNA DNA primase: synthesis of RNA Topoisomerase: transient DNA breaks DNA polymerase III: extend the RNA primers (deoyxribonucleotide). It is holoenzymes © John Wiley & Sons, Inc.
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DNA Replication Synthesis of the leading strand is continuous.
Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together. © John Wiley & Sons, Inc.
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The E. coli Replisome Replisome: complete replication apparatus
© John Wiley & Sons, Inc.
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Rolling-Circle Replication
Replication’s Models- O-shape Eye-shape Y-shape Rolling-circle (viruses, bacteria , amphibians) _______________________________________ 1- Nick by specific endonucleases 2-parental DNA is intact and functions as template 3-DNA polymerase 5’ to 3’ 4- displacement of one of the DNA strand © John Wiley & Sons, Inc.
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DNA replication is complex, requiring the participation of a large number of proteins.
DNA synthesis is continuous on the progeny strand that is being extended in the overall 5'3' direction, but is discontinuous on the strand growing in the overall 3'5' direction. © John Wiley & Sons, Inc.
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DNA synthesis is catalyzed by enzymes called DNA polymerases.
New DNA chains are initiated by short RNA primers synthesized by DNA primase. DNA synthesis is catalyzed by enzymes called DNA polymerases. All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied. © John Wiley & Sons, Inc.
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All DNA polymerases have an absolute requirement for a free 3’-OH on the primer strand, and all DNA synthesis occurs in the 5’ to 3’ direction. The 3’ to 5’ exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired (match) nucleotides at the 3’ termini of primer strands. © John Wiley & Sons, Inc.
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The enzymes and DNA-binding proteins involved in replication assembled into a replisome at each replication fork and act in concert as the fork moves along the parental DNA molecule. © John Wiley & Sons, Inc.
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Unique Aspects of Eukaryotic Chromosome Replication
Although the main features of DNA replication are the same in all organisms, some processes occur only in eukaryotes. Viruses and E.coli © John Wiley & Sons, Inc.
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DNA Replication in Eukaryotes
Shorter RNA primers and Okazaki fragments DNA replication only during S phase (bacteria will duplicate DNA only in a rich environment) Multiple origins of replication (bacteria shows one origins of replication) Nucleosomes (nucleosomes are not present in bacteria) Telomeres (telomeres are not present in bacteria) Cell Cycle --check points ----S phase ----Mitosis © John Wiley & Sons, Inc.
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Bidirectional Replication from Multiple Origins in Eukaryotes
Pulse chase experiments with 3H-thymidine ---Origins of replication ---Large number of replicons (1 vs ~1x105) © John Wiley & Sons, Inc.
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Replicon: segment of DNA containing one Origin (O) and Two termini (T)
© John Wiley & Sons, Inc.
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The Eukaryotic Replisome
SV40 virus: DNA virus (histones) Bacteria replication --unwind parental DNA (without histones) ----DNA helicase ----Topoisomerase ----Single -strand DNA binding protein ----DNA polymerase III © John Wiley & Sons, Inc.
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Eukaryotic Replication Proteins
----parental DNA (with histones) ----Polymerases () Pol initiation of replication (origins) priming of Okazaki fragment complex with DNA primase Pol synthesis of lagging strand Pol synthesis of leading strand ----accessories proteins: PCNA and Rf-C (sliding clamp) ----Pol have exonuclease activity ( 3’to 5”) =proofreading ----Other Pols (pie, lambda, phi, rho, and mu) do not have exonuclease activity ( 5’to 3”) ----Ribonulceases H1 and FEN-1 Produce the RNA/DNA chain Proliferating Cell Nuclear Antigen: PCNA © John Wiley & Sons, Inc.
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Nucleosome Spacing in Replicating Chromatin
Assembly and disassembly of nucleosomes © John Wiley & Sons, Inc.
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Chromatin can have alternative states
Inactive--DNA/histones Active--Polymerase/TFs Polymaerase/TFs NO TRANSCRIPTION HISTONES TRANSCRIPTION “The addition of either TFs or nucleosomes may form stable structures that can not be changed by modifying the equilibrium with free components” How is the chromatin structure regulated?
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Chromatin remodeling © John Wiley & Sons, Inc.
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The Telomere “extension” Problem
DNA polymerase can not replicate the terminal DNA ---too big ---not enough space ( 3’-OH, primer) © John Wiley & Sons, Inc.
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Telomerase (Reverse Transcriptase) G-rich telomere sequence 5’ to 3’
Aging (early aging….progerias) Immortality: Cancer and Normal cells Senescence: normal diploid cells cease to divide, (about 45 to 50 cell divisions). © John Wiley & Sons, Inc.
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Telomere Length and Aging
Shorter telomeres are associated with cellular senescence and death. Diseases causing premature aging are associated with short telomeres. Apoptosis (self-destruction): programmed cell death © John Wiley & Sons, Inc.
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Telomeres Are Essential for Survival
Figure 28.32
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Dna polymerases classification as follows:
Prokaryotic DNA polymerases Pol I to V Eukaryotic DNA polymerases Pol theta, pie, lambda, phi, rho, and mu. Based on sequence homology A, B, C, D, X, Y, and RT bacterial © John Wiley & Sons, Inc.
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Since the parental double helix must rotate 360° to unwind each gyre of the helix,
during the semi-conservative replication of the bacterial chromosome, some kind of “swivel” must exist. What do geneticists now know that the required swivel is? a) Topoisomerase b) Helicase c) A transient single-strand break produced by the action of topoisomerases d) A transient single-strand break produced by the action of helicases e) A transient single-strand break produced by the action of Ligase In the E. coli chromosome the origin of replication, called oriC, is characterized as being rich in: a) A-G base pairs b) A-C base pairs c) C-G base pairs d) C-T base pairs e) None of the above
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Telomere length has not been correlated with:
a) Aging b) Sex determination c) Progeria d) Cancer e) All of these
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