Structure and Replication of DNA ... DNA Replication: Semiconservative Replication- DNA unzips and a

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  • Structure and Replication of DNA

  • John Kyrk Animations

    • http://www.johnkyrk.com/DNAanatomy.html

    http://www.johnkyrk.com/DNAanatomy.html

  • Are Genes Composed of DNA or Protein?

    • DNA

    – Only four nucleotides

    • thought to have monotonous structure

    • Protein

    – 20 different amino acids – greater potential variation

    – More protein in chromosomes than DNA

  • Bacterial Transformation Experiments

    Fredrick Griffith (1928) –demonstrate the existence of “Transforming Principle,” a substance able to transfer a heritable phenotype (trait) from one strain of bacteria to another.

    Avery MacLeod and McCarty – determine the

    transforming principle was DNA.

  • Streptococcus Pneumoniae

  • Griffith Experiment

  • Avery Experiment

  • Viruses Injecting DNA into a Bacterium

    Bacterial cell

    Phage head

    Tail sheath

    Tail fiber

    DNA

    1 0

    0 n

    m

  • Hershey Chase Experiment – Viruses can be used to transfer traits and therefore DNA

  • Traits can be transferred if DNA is transferred.

    (a) Tobacco plant expressing a firefly gene

    (b) Pig expressing a jellyfish gene

  • Additional Evidence • Chargaff Ratios

    • % A = %T and %G = %C (Complexity in DNA Structure)

    A T G C

    Arabidopsis 29% 29% 20% 20%

    Humans 31% 31% 18% 18%

    Staphlococcus 13% 13% 37% 37%

    • DNA Content of Diploid and Haploid cells – Haploid cells contain half of the amount of DNA

    Gametes Somatic Cells Humans 3.25pg 7.30 pg

    Chicken 1.267pg 2.49pg

  • DNA

    Friedrich Meischer (1869) extracted a phosphorous rich material from nuclei of which he named nuclein

    DNA – deoxyribonucleic acid - Monomer – Nucleotide

    Deoxyribose Phosphate Nitrogenous Base (4 types – 2

    purines G & A; 2 pyrimidines T & C)

    - Phosphodiester Bond linkage - DNA has direction - 5’ and 3’ ends - Chromosomes are composed of DNA

  • Fig. 16-UN1

    Purines have two rings. Pyrimidines have one ring.

    Purine + purine: too wide

    Pyrimidine + pyrimidine: too narrow

    Purine + pyrimidine: width consistent with X-ray data

  • Watson and Crick Model • Franklins X-Ray Data

    – DNA is Double Helix • 2 nm diameter

    • Phosphates on outside

    • 3.4 nm periodicity

    • Bases 0.34nm apart

    • Watson and Crick

    – Base Pairing- Purine with Pyrimidine (A/T & C/G)

  • DNA double helix (2 nm in diameter)

    Nucleosome (10 nm in diameter)

    Histones Histone tail

    H1

    DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

    DNA Structure – Chromatin = unwound DNA

    video

    http://www.myteacherpages.com/webpages/BMorton/files/Media/1621DNAPacking_A.swf

  • Chromatin coils around proteins to form Chromosomes

    30-nm fiber

    Chromatid (700 nm)

    Loops Scaffold

    300-nm fiber

    Replicated chromosome (1,400 nm)

    30-nm fiber Looped domains (300-nm fiber)

    Metaphase chromosome

  • 30 nm chromatin fiber

    1. Held together by histone tails interacting with neighboring nucleosomes 2. Inhibits transcription 3. Allows DNA replication

  • DNA Replication:

    Semiconservative Replication- DNA unzips and a new strand builds on the inside. The new strands each have a

    piece of the “old” DNA

  • Other Models of Replication

    Conservative Replication

    Semi-Conservative Replication

    Dispersive Replication

  • Culture Bacteria in 15N isotope (DNA fully 15N)

    One Cell Division in 14N

    2nd Cell Division in 14N

    Less Dense More Dense

    Density Centrifugation

    15N DNA 15N/14N DNA

    15N/14N DNA

    14N DNA

  • DNA Replication: A Closer Look

    • The copying of DNA is remarkable in its speed and accuracy

    • More than a dozen enzymes and other proteins participate in DNA replication

    Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

  • • Replication bubbles are the “unzipped” sections where replication occurs all along the molecule

    • At the end of each replication bubble is a replication fork: a Y-shaped region where new DNA strands are elongating

    • Helicase: enzyme that unzips the double helix at the replication forks

    • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template

    • Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

    Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

  • Origins of Replication Video

    http://www.myteacherpages.com/webpages/BMorton/files/Media/1612OriginsOfReplicationA.swf

  • Fig. 16-13

    Topoisomerase

    Helicase

    Primase Single-strand binding

    proteins

    RNA

    primer

    5 5

    5 3

    3

    3

  • DNA Polymerase – enzyme that builds the new strand

    3’ 5’

    3’ 5’ Pol

  • Pol

    Leading and Lagging Strands – Polymerase only works on the 3’ to 5’ DNA side. Must do the 5’ to 3’ side in

    segments called Okazaki fragments. 3’ to 5’ = Leading (easy) strand; 5’ to 3’ = lagging (segmented) strand

    5’

    5’

    3’

    3’

    Leading Strand

    Lagging Strand

    Pol

    3’

    5’

    RNA Primer

    Video

    http://www.myteacherpages.com/webpages/BMorton/files/Media/1615LeadingStrandA.swf

  • Other Proteins at Replication Fork

    Pol

    5’

    5’

    3’

    3’

    Leading Strand

    Lagging Strand

    Pol

    3’

    5’

    Helicase

    Single Stranded Binding Proteins

    Primase

    DNA Pol I

    Ligase

    DNA Pol III

  • Lagging strand

    assembly and

    Okazaki

    fragments

    Overview

    Origin of replication

    Leading strand

    Leading strand

    Lagging strand

    Lagging strand

    Overall directions

    of replication

    Template

    strand

    RNA primer

    Okazaki

    fragment

    Overall direction of replication

    1 2

    3

    2

    1

    1

    1

    1

    2

    2

    5

    1 3

    3

    3

    3

    3

    3

    3

    3

    3

    5

    5

    5

    5

    5

    5

    5

    5

    5

    5

    5 3

    3

  • Damaged DNA Nuclease Excision Repair – cut and replace

    Nuclease

    DNA Polymerase

    Ligase

  • Replicating the Ends of DNA Molecules

    • Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes

    • The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules

    Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

  • Replicating Ends of Linear Chromosomes

  • Fig. 16-19

    Ends of parental

    DNA strands Leading strand

    Lagging strand

    Lagging strand

    Last fragment Previous fragment

    Parental strand

    RNA primer

    Removal of primers and

    replacement with DNA

    where a 3 end is available

    Second round

    of replication

    New leading strand

    New lagging strand

    Further rounds

    of replication

    Shorter and shorter daughter molecules

    5

    3

    3

    3

    3

    3

    5

    5

    5

    5

  • • If chromosomes of germ (sex) cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce

    • An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells; it adds temporary DNA so the strand can be completed

    Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

  • Telomerase

  • Telomeres

    1 µm

  • END STRUCTURE/REPLICATION

    • Crash Course Video

    • DNA Activities

  • Chapter 10 From Gene to Protein

  • Protein Synthesis: overview

     One gene-one enzyme hypothesis (Beadle and Tatu

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