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					  Chapter 12: DNA

       Mrs. Lipenta
Upper Moreland High School
What is DNA?
   DNA stands for
    deoxyribonucleic acid.
   It was discovered by
    James Watson and
    Francis Crick
   The shape of DNA can
    be described as a
    double helix.
    ◦ Think about it as a
      spiral staircase, or a
      ladder.
Watson and Crick
   DNA is composed of nucleotides
   Each nucleotide is made up of 3 parts; a
    sugar, a phosphate, and a nitogenous base.




 In DNA the         P
   sugar is
deoxyribose.
                           S      N.B.
   The sides of the
    ladder (called the
    DNA backbone)
    is composed of
    the sugar and
    phosphates.
   The rungs of the
    ladder contain
    the nitrogenous
    bases.
Chargaff            1947
    DNA composition: “Chargaff’s rules”
     ◦ varies from species to species
         humans:
                A = 30.9%
                 T = 29.4%
                G = 19.9%
                C = 19.8%

            The bases pair up based
              on Chargoff’s Rules
                 A=T & C=G
   A = Adenine
   T = Thymine
   C = Cytosine
   G = Guanine
   Pairing like nucleotides did not fit the
    uniform diameter indicated by the X-ray data.
    ◦ A purine-purine pair would be too wide and a
      pyrimidine-pyrimidine pairing would be too short.
    ◦ Only a pyrimidine- purine pairing would produce
      the 2-nm diameter indicated by the X-ray data.
   How does it fit together?
   We need to look at the numbered Carbons

   Pay close attention to
    the 3’ and 5’ carbons

   5’ attaches to the P
   3’ attaches to the
    next nucleotide
   Remember nucleotides are monomers
   How do we link monomers?
   Through bonding
   But where do we get the energy?

                                        We need
                                        energy!
   What carrier energy from place to place in the
    body?
   Show me the Money!!!!

   Yes it is our old friend ATP…

   And he has brought
    along some friends
             CTP

      GTP

                   Notice
                  anything?

ATP
            TTP
Nucleotides

                    CTP

        GTP

                    WOW
                    Why?
  ATP
              TTP
           Energy



B.Y.O.E.
   Process where we end up with 2 exact copies
    of DNA.
   Steps of Replication
    ◦ Step 1 – Unzip DNA
    ◦ Step 2 – Add free nucleotides to the original strands
      of DNA. These nucleotides will add together and
      form the new strand of DNA.
    ◦ Step 3 – End up with 2 exact copies
Replication: 1st step
    Unwind DNA
     ◦ helicase enzyme
        unwinds part of DNA helix
        stabilized by single-stranded binding proteins




     single-stranded binding proteins
    Replication                             5'                          3'


    Step 2
                                         energy
                                            DNA
                                            P III

     Adding bases                       energy
      ◦ can only add nucleotides
        to 3 end of a growing
        DNA strand
                                         energy
      ◦ strand grow 5'3’


                                         energy




                                            3'                          5'
                                           leading strand   2005-2006
Thanks to Kim Foglia for the animation
   New nucleotides can only fit one way.
    Remember they bring their energy with them.

   One side is copied continuously… no problem
    there.

   What about the other side?
          Thanks to Kim Foglia for the animation


     5'                                     3'              5'                            3'




                                             ligase




                                                   energy
                                                            3'
3'                                                          leading strand           5'
     lagging strand 5'                                                       2005-2006
Copying DNA
    Replication of DNA
     ◦ base pairing allows
       each strand to serve
       as a pattern for a
       new strand
Leading & Lagging strands
Leading strand
 - continuous
 synthesis


                                 Okazaki




                 Lagging strand
                   - Okazaki fragments
                   - joined by ligase
                    - “spot welder” enzyme
                                2005-2006
Okazaki fragments
   Helicase comes in and unzips the DNA
   Single-Stranded Bonding Proteins hold it
    open
   One side – the leading strand gets copied
    continuously
   The other side – the lagging strand gets
    copied in little fragments called Okazaki
    fragments
   DNA polymerase III is responsible for adding
    the nucleotides to the DNA, but it can only
    add to the strand, it can not start copying.
   Primase starts the copying by adding a small
    amount of RNA to the DNA. That small
    section of RNA is called the Primer
Priming
DNA
synthesis
   We can’t have
    RNA in our DNA…
   DNA polymerase I
    comes in and
    removes sections
    of RNA primer
    and replaces with
    DNA nucleotides
   Also proofreads
    and corrects the
    DNA
   Now we have little fragments of DNA, need to
    glue them together.
   This is a job for Ligase.
   Ligase acts like the glue and will link the
    Okazaki fragments together.
Replication fork
               DNA
               polymerase III     lagging strand
DNA
polymerase I
                      Okazaki                                  3’
                                               primase
                      fragments                                     5’
5’     ligase
 3’                                        5’     SSB

                                                3’        helicase
                                         DNA
                                         polymerase III
5’
      leading strand
 3’
                    direction of replication
You need to make…
   helicase
   DNA polymerase III
   primase
   DNA polymerase I
   ligase
   single-stranded binding proteins
   Show me DNA
    replication
   Also don’t forget to
    B.Y.O.E.
   If I asked you a question about something in
    the textbook, would you read the entire
    textbook?

   What would you do?

   Why?
   What is the complementary strand?
   DNA = ATCGCGCTAATCGATCG
   DNA = ?


                         Chargoff’s
                          Rules
   During what part of the
    cell cycle does DNA
    replication occur?
And in the end…
   Ends of
    chromosomes are
    eroded with each
    replication
    ◦ an issue in aging?
    ◦ ends of
      chromosomes are
      protected by
      telomeres




                           2005-2006
Telomeres
   Expendable,
    non-coding
    sequences at ends of
    DNA
    ◦ short sequence of bases
      repeated 1000s times
    ◦ TTAGGG in humans
   Telomerase enzyme in
    certain cells
    ◦ enzyme extends
      telomeres
    ◦ prevalent in cancers
        Why?


                                2005-2006
Editing & proofreading DNA
     1000 bases/second =
      lots of typos!
     DNA polymerase I
      ◦ proofreads & corrects
        typos
      ◦ repairs mismatched bases
      ◦ excises abnormal bases
          repairs damage
           throughout life
      ◦ reduces error rate from
        1 in 10,000 to
        1 in 100 million bases
Fast & accurate!
    It takes E. coli <1 hour to copy
     5 million base pairs in its single
     chromosome
     ◦ divide to form 2 identical daughter cells
    Human cell copies its 6 billion bases &
     divide into daughter cells in only few
     hours
     ◦ remarkably accurate
     ◦ only ~1 error per 100 million bases
     ◦ ~30 errors per cell cycle
DNA Replication                        A Team Effort!
    Large team of enzymes coordinates replication
   Bonding in DNA
                                    hydrogen

                          5’        bonds
                                                      3’


    phosphodiester

    bonds



                          3’
                                                      5’

….strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?
   The march to understanding that DNA
    is the genetic material
    ◦ T.H. Morgan (1908)
    ◦ Frederick Griffith (1928)
    ◦ Avery, McCarty & MacLeod (1944)
    ◦ Hershey & Chase (1952)
    ◦ Watson & Crick (1953)
    ◦ Meselson & Stahl (1958)
                                    1908 | 1933


   T.H. Morgan
    ◦ working with Drosophila (fruit
      flies)
    ◦ genes are on chromosomes
    ◦ but is it the protein or the DNA
      of the chromosomes that are
      the genes?
       through 1940 proteins
        were thought to be
        genetic material… Why?

          What’s so impressive
          about proteins?!
                                         1928
The “Transforming Factor”
    Frederick Griffith
     ◦ Streptococcus pneumonia
       bacteria
        was working to find cure for
         pneumonia
     ◦ harmless live bacteria mixed
       with heat-killed infectious
       bacteria causes disease in mice
     ◦ substance passed from dead
       bacteria to live bacteria =
       “Transforming Factor”
Transformation?
something in heat-killed bacteria could still
transmit disease-causing properties
 The “Transforming Factor”
                                                            mix heat-killed
                                                            pathogenic &
live pathogenic    live non-pathogenic heat-killed          non-pathogenic
strain of bacteria strain of bacteria  pathogenic bacteria bacteria
 A.                B.                C.              D.




mice die           mice live           mice live          mice die
                                              1944
DNA is the “Transforming Factor”
    Avery, McCarty & MacLeod
     ◦ purified both DNA & proteins from
       Streptococcus pneumonia bacteria
        which will transform non-pathogenic
         bacteria?
     ◦ injected protein into bacteria
        no effect                    What’s the
                                      conclusion?
     ◦ injected DNA into bacteria
        transformed harmless bacteria
         into virulent bacteria
Avery, McCarty & MacLeod




Oswald Avery
                                Colin MacLeod

               Maclyn McCarty
                                        1952 | 1969


   Hershey & Chase
    ◦ classic “blender” experiment
    ◦ worked with bacteriophage
       viruses that infect bacteria
    ◦ grew phage viruses in 2 media, radioactively
      labeled with either
       35S in their proteins
       32P in their DNA
    ◦ infected bacteria with
      labeled phages
Models of DNA Replication
    Alternative models
     ◦ so how is DNA copied?
Semi-conservative replication
   Meselson & Stahl
    ◦ label nucleotides of “parent” DNA strands with
      heavy nitrogen = 15N
    ◦ label new nucleotides with lighter isotope = 14N

                    “The Most Beautiful Experiment in Biology ”



     How to
     prove it!
                                             1958
Semi-conservative replication
   Make predictions…
    ◦ 15N strands replicated in 14N medium
    ◦ 1st round of replication?
    ◦ 2nd round?
Hershey & Chase




    Martha Chase   Alfred Hershey
                   Protein coat labeled               DNA labeled with   32P
                   with 35S
Hershey                                T2 bacteriophages
                                       are labeled with

& Chase                                radioactive isotopes
                                       S vs. P

                                       bacteriophages infect
                                       bacterial cells




                                   bacterial cells are agitated
Which                              to remove viral protein coats
radioactive
marker is found
inside the cell?

Which molecule
carries viral                                      32P
                         35Sradioactivity              radioactivity found
genetic info?            found in the medium       in the bacterial cells
                                                                     2005-2006
   Radioactive phage & bacteria in
    blender
    ◦ 35S phage
       radioactive proteins stayed in
        supernatant
       therefore protein did NOT enter bacteria
    ◦ 32P phage
       radioactive DNA stayed in pellet
       therefore DNA did enter bacteria
    ◦ Confirmed DNA is “transforming factor”
Your
DNA!

				
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posted:2/27/2012
language:English
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