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DNA Replication Lecture - ... DNA Replication D. DNA Primers DNA polymerases cannot initiate synthesis of a polynucleotide because they can only add nucleotides to the end of an existing

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Text of DNA Replication Lecture - ... DNA Replication D. DNA Primers DNA polymerases cannot initiate...

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    �� Chapter 16~Chapter 16~The Molecular Basis of InheritanceThe Molecular Basis of Inheritance

    DNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA Structure

    �� ChargaffChargaff ratio of nucleotide ratio of nucleotide bases (A=T; C=G)bases (A=T; C=G)

    �� Watson & Crick Watson & Crick (Wilkins, Franklin)(Wilkins, Franklin)

    �� The Double Helix The Double Helix √√ nucleotidesnucleotides: :

    nitrogenous base (thymine, nitrogenous base (thymine, adenine, cytosine, guanine); adenine, cytosine, guanine); sugar deoxyribose; sugar deoxyribose; phosphate groupphosphate group

    DNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA Structure DNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA Bonding

    �� Purines: Purines: ‘‘AA’’ & & ‘‘GG’’

    �� Pyrimidines: Pyrimidines: ‘‘CC’’ & & ‘‘TT’’ (Chargaff rules)(Chargaff rules)

    �� ‘‘AA’’ H+ bonds (2) with H+ bonds (2) with ‘‘TT’’ and and ‘‘CC’’ H+ bonds (3) H+ bonds (3) with with ‘‘GG’’

    �� Van der Waals Van der Waals attractions between the attractions between the stacked pairsstacked pairs

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    DNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTURE 1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication 1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication

    A. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. Overview �� In 2In 2ndnd paper Watson and Crick published hypothesis for how paper Watson and Crick published hypothesis for how

    DNA replicates.DNA replicates.

    �� Because each strand is complementary, each can form a Because each strand is complementary, each can form a template when separated.template when separated.

    �� When a cell copies a DNA molecule, each strand serves as a When a cell copies a DNA molecule, each strand serves as a templatetemplate for ordering nucleotides into a new complementary for ordering nucleotides into a new complementary strand.strand.

    �� Nucleotides line up along the template strand according to the Nucleotides line up along the template strand according to the basebase--pairing rulespairing rules..

    �� The nucleotides are linked to form new strands.The nucleotides are linked to form new strands.

    DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

    �� Watson and Crick: Watson and Crick: semiconservativesemiconservative replication, a replication, a double helix replicates, each of double helix replicates, each of the daughter molecules will have the daughter molecules will have 1 old strand and 1 new strand.1 old strand and 1 new strand.

    �� Other models: Other models: conservativeconservative and and dispersivedispersive

    �� MeselsonMeselson and Stahl supported and Stahl supported the the semiconservativesemiconservative modelmodel, , proposed by Watson and Crick, proposed by Watson and Crick, over the other two models:over the other two models:

    DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

    1.1. Labeled the nucleotides of the old strands with Labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N), while any a heavy isotope of nitrogen (15N), while any new nucleotides were indicated by a lighter new nucleotides were indicated by a lighter isotope (14N).isotope (14N).

    2.2. Replicated strands could be separated by Replicated strands could be separated by density in a centrifuge.density in a centrifuge.

    3.3. Each modelEach model—— the semithe semi--conservative model, conservative model, the conservative model, and the dispersive the conservative model, and the dispersive modelmodel—— made specific predictions made specific predictions on the on the density of replicated DNA strandsdensity of replicated DNA strands..

    4.4. The first replication in the 14N medium The first replication in the 14N medium produced a band of hybrid (15Nproduced a band of hybrid (15N--14N) DNA, 14N) DNA, eliminating the conservative modeleliminating the conservative model..

    5.5. A second replication produced both light and A second replication produced both light and hybrid DNA, eliminating the dispersive model hybrid DNA, eliminating the dispersive model and supporting the and supporting the semiconservativesemiconservative model.model.

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    DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication: B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

    DNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA Replication

    2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication

    In bacteriaIn bacteria, this is a single specific sequence of nucleotides that is reco, this is a single specific sequence of nucleotides that is recognized by the gnized by the replication enzymes.replication enzymes.

    �� These enzymes separate the strands, forming a These enzymes separate the strands, forming a replication replication ““bubblebubble””..

    In eukaryotesIn eukaryotes, there may be hundreds or thousands of origin sites per chromos, there may be hundreds or thousands of origin sites per chromosomeome

    �� HelicaseHelicase is the enzyme that catalyzes the untwisting of DNA at the repliis the enzyme that catalyzes the untwisting of DNA at the replication forkcation fork

    �� Replication forksReplication forks: the Y: the Y--shaped region where new DNA is elongating.shaped region where new DNA is elongating.

    �� The replication bubbles elongate as the DNA is replicated and evThe replication bubbles elongate as the DNA is replicated and eventually fuse entually fuse

    2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication

    B. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNA

    �� DNADNA polymerasespolymerases catalyze the elongation of catalyze the elongation of new DNA at a replication fork.new DNA at a replication fork.

    �� As nucleotides align with complementary As nucleotides align with complementary bases along the template strand, they are bases along the template strand, they are added to the growing end of the new strand by added to the growing end of the new strand by the polymerase.the polymerase.

    �� The rate of elongation is about 500 nucleotides The rate of elongation is about 500 nucleotides per second in bacteria and per second in bacteria and 50 per second50 per second in in human cells. human cells.

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    2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication

    B. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNA

    �� Raw nucleotides = nucleoside Raw nucleotides = nucleoside triphosphatestriphosphates: : nitrogen base, nitrogen base, deoxyribosedeoxyribose, , triphosphatetriphosphate tail.tail.

    �� As each nucleotide is added, the last two As each nucleotide is added, the last two phosphate groups are hydrolyzed to form phosphate groups are hydrolyzed to form pyrophosphatepyrophosphate..

    �� The The exergonicexergonic hydrolysis hydrolysis of pyrophosphate to of pyrophosphate to two two inorganic phosphateinorganic phosphate molecules drives molecules drives the polymerization of the polymerization of the nucleotide to the the nucleotide to the new strand.new strand.

    2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication

    C. C. C. C. C. C. C. C. AntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallel Nature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNA

    �� The sugarThe sugar--phosphate backbones of phosphate backbones of the double helix run in o

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