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					Chapter 16~      The Molecular Basis of
              Inheritance
        Scientific History
• The march to understanding that DNA is
  the genetic material
  – T.H. Morgan (1908)
  – Frederick Griffith (1928)
  – Avery, McCarty & MacLeod (1944)
  – Erwin Chargaff (1947)
  – Hershey & Chase (1952)
  – Watson & Crick (1953)
  – Meselson & Stahl (1958)
 The “Transforming                               1928
                                        Principle”
• Frederick Griffith
  – Streptococcus pneumonia bacteria
     • was working to find cure for pneumonia
  – harmless live bacteria (“rough”) mixed
    with heat-killed pathogenic bacteria
    (“smooth”) causes fatal disease in
    mice
  – a substance passed from dead
    bacteria to live bacteria to change
    their phenotype
     • “Transforming Principle”
         The “Transforming Principle”
                                 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




   Transformation = change in phenotype
   something in heat-killed bacteria could still transmit
   disease-causing properties
     DNA is the “Transforming
                              1944
             Principle”
• Avery, McCarty & MacLeod
  – purified both DNA & proteins separately from
    Streptococcus pneumonia bacteria
     • which will transform non-pathogenic bacteria?
  – injected protein into bacteria
     • no effect
  – injected DNA into bacteria
     • transformed harmless bacteria into
       virulent bacteria

                                                  mice die
Avery Experiment
  Avery, McCarty & MacLeod
• Conclusion
  – first experimental evidence that DNA was the genetic
    material




Oswald Avery     Maclyn McCarty         Colin MacLeod
                                        1952
        Confirmation of DNA
• 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
                   Protein coat labeled                DNA labeled with 32P
                   with 35S
Hershey                                   T2 bacteriophages
                                          are labeled with
   &                                      radioactive isotopes
                                          S vs. P

 Chase                                    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            35S                          32P
                            radioactivity                 radioactivity found
genetic info?            found in the medium          in the bacterial cells
      Blender experiment
• Radioactive phage & bacteria in blender
  – 35S phage
    • radioactive proteins stayed in supernatant
    • therefore viral protein did NOT enter bacteria
  – 32P phage
    • radioactive DNA stayed in pellet
    • therefore viral DNA did enter bacteria
  – Confirmed DNA is “transforming factor”
                                1952
  Hershey & Chase




Martha Chase   Alfred Hershey
                   Chargaff                 1947

• DNA composition: “Chargaff’s rules”
  – varies from species to species
  – all 4 bases not in equal quantity
  – bases present in characteristic ratio
    • humans:
                A = 30.9%
                T = 29.4%
                G = 19.9%
                C = 19.8%
         Structure of DNA                         1953

• Watson & Crick
  – developed double helix model of DNA
    • other leading scientists working on question:
       – Rosalind Franklin
       – Maurice Wilkins
       – Linus Pauling




     Franklin             Wilkins              Pauling
                           1953 article in Nature
Watson and Crick




Watson             Crick
Rosalind Franklin (1920-1958)
      Double helix structure of DNA




“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.”                                                Watson & Crick
      Directionality of DNA
• You need to      PO4               nucleotide
  number the
  carbons!
  – it matters!                        N base

                  5 CH2
                                 O

                  4         ribose        1


                       3             2
                            OH
                                                  5
The DNA backbone
                                   PO4
• Putting the DNA
                                                       base
  backbone together               5 CH2

  – refer to the 3 and 5                   O
                                   4                   1
    ends of the DNA                      C
                                        3         2
     • the last trailing carbon       O
                                  –O P O

                                      O                 base
                                    5 CH2
                                                   O
                                        4                   1

                                             3         2
                                              OH
                                                  3
Anti-parallel strands
• Nucleotides in DNA
  backbone are bonded from
  phosphate to sugar between       5   3
  3 & 5 carbons
  – DNA molecule has “direction”
  – complementary strand runs in
    opposite direction




                                   3   5
                  Bonding in DNA
                                     hydrogen

                                     bonds
                          5                          3

    covalent
    phosphodiester

    bonds


                          3
                                                      5

….strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?
      Base pairing in DNA
• Purines
  – adenine (A)
  – guanine (G)
• Pyrimidines
  – thymine (T)
  – cytosine (C)
• Pairing
  –A:T
    • 2 bonds
  –C:G
    • 3 bonds
            But how is DNA copied?
     • Replication of DNA
         – base pairing suggests that
           it will allow each side to
           serve as a template for a
           new strand




“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.”                                              — Watson & Crick
            Copying DNA
• Replication of DNA
  – new strand is 1/2 parent
    template &
    1/2 new DNA
    • semi-conservative
      copy process
Semiconservative replication,
• when a double helix replicates each of the daughter molecules will
  have one old strand and one newly made strand.
• Experiments in the late 1950s by Matthew Meselson and Franklin
  Stahl supported the semiconservative model, proposed by Watson
  and Crick, over the other two models. (Conservative & dispersive)
          Interesting Facts
• E. Coli takes 25 minutes to copy its 5
  million base pairs and make its daughter
  cells


• Human cells copy 6 billion base pairs and
  duplicate in a few hours
• There is only @ one error per 10 billion
  base pairs
    DNA Replication
• Large team of enzymes coordinates replication
           Replication: 1st step
 • Unwind DNA
     – helicase enzyme
         • unwinds part of DNA helix
         • stabilized by single-stranded binding proteins
                                        helicase




single-stranded binding proteins        replication fork
      Replication: 2nd step
                  Build daughter DNA
                  strand
                    add new
                     complementary bases
                    DNA polymerase III




DNA
Polymerase III
                            5                3
                   Replication
                         energy
                             DNA
• Adding bases               Polymerase III

  – can only add          energy
    nucleotides to           DNA
                             Polymerase III
    3 end of a growing
    DNA strand            energy
                             DNA
    • need a “starter”       Polymerase III
      nucleotide to
      bond to             energy
                             DNA
  – strand only grows        Polymerase III
    53
                            3                5
            Leading & Lagging strands
Limits of DNA polymerase III
    can only build onto 3 end of an
     existing DNA strand                                                              5




    3                                 5
                                              3
                                                   5
                                                                 3   5        5
                                                                                     3

                                                                           Lagging strand
                                                        ligase
                 growing          3
                 replication fork
    5
                                                                           Leading strand



Lagging strand
                                                                                 
                                                                                3    5


                                                                                      3

    Okazaki fragments                      DNA polymerase III
    joined by ligase                              Leading strand
          “spot welder” enzyme                            continuous synthesis
          Replication fork / Replication
     3              bubble                                                                                      5

     5                                                                                                          3

                         DNA polymerase III
                                                                       leading strand
                                                          5
     3                                                              3                                          5
                                                                5        5
     5                                              3                                                          3
                                                                          lagging strand



                                                3   5
                                       5
3                                 lagging strand    leading strand
                        5                                                                      growing
                                                                                     3         replication fork 5
5         growing
           replication fork                                                                5
                                   leading strand                                                                3
                                                               lagging strand
                              3
                                                                               5
                                                               5 5
          Starting DNA synthesis: RNA
                    primers
Limits of DNA polymerase III
    can only build onto 3 end of an
     existing DNA strand                                                 5


                                                         3     5       3
                                                 5
                                            3
    3                                5

                growing          3                                   primase
                replication fork           DNA polymerase III
    5

                                                                     RNA 5


RNA primer                                                               3

    built by primase
    serves as starter sequence for DNA
     polymerase III
         Replacing RNA primers with DNA
DNA polymerase I
    removes sections of RNA primer and
                                      DNA polymerase I
     replaces with DNA nucleotides                             5

                                                               3


    3
                                5             ligase
               growing          3
               replication fork
    5

                                                         RNA   5


                                                               3

But DNA polymerase I still
can only build onto 3 end of
an existing DNA strand
                   Chromosome erosion
All DNA polymerases can
only add to 3 end of an                 DNA polymerase I
existing DNA strand                                                 5

                                                                    3


    3
                                5
               growing          3
               replication fork          DNA polymerase III
    5

                                                              RNA   5


Loss of bases at 5 ends                                            3

in every replication
    chromosomes get shorter with each replication
    limit to number of cell divisions?
                                       Telomeres
Repeating, non-coding sequences at the end
of chromosomes = protective cap
    limit to ~50 cell divisions                                      5

                                                                      3


    3
                                  5
                 growing          3                            telomerase
                 replication fork
    5

                                                                      5


Telomerase                                            TTAAGGG TTAAGGG 3
    enzyme extends telomeres
    can add DNA bases at 5 end
    different level of activity in different cells
          high in stem cells & cancers -- Why?
                   Replication fork
               DNA
               polymerase III       lagging strand
DNA
polymerase I
                                                                 3’
                      Okazaki                   primase
                      fragments                                       5’
 5’       ligase
   3’                                          5’    SSB

                                                    3’     helicase

                                         DNA
                                         polymerase III
5’      leading strand
 3’
                    direction of replication
                                SSB = single-stranded binding proteins
                DNA polymerases
    • DNA polymerase III
        – 1000 bases/second!                 Roger Kornberg
                                             2006
        – main DNA builder
    • DNA polymerase I
        – 20 bases/second
        – editing, repair & primer removal
DNA polymerase III                           Arthur Kornberg
enzyme                                       1959
  Editing & proofreading DNA
• 1000 bases/second =
  lots of typos!
• DNA polymerase I
  – proofreads & corrects typos
  – repairs mismatched bases
  – removes abnormal bases
     • repairs damage
       throughout life
  – reduces error rate from
    1 in 10,000 to
    1 in 100 million bases
  Nucleosomes                              8 histone
                                           molecules
• “Beads on a string”
  – 1st level of DNA packing
  – histone proteins
    • 8 protein molecules
    • positively charged amino acids
    • bind tightly to negatively charged
      DNA
  DNA packing as gene
       control
• Degree of packing of DNA regulates transcription
  – tightly wrapped around histones
     • no transcription
     • genes turned off    heterochromatin
                            darker DNA (H) = tightly packed
                           euchromatin
                            lighter DNA (E) = loosely packed

                                      H     E

				
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