Chapter 16 by yurtgc548

VIEWS: 3 PAGES: 49

									      Chapter 16.
                    DNA
             The Genetic Material
                 Replication



AP Biology
   Scientific History
    § The march to understanding that DNA is
        the genetic material
         u T.H. Morgan (1908)
         u Frederick Griffith (1928)

         u Avery, McCarty & MacLeod (1944)

         u Hershey & Chase (1952)

         u Watson & Crick (1953)

         u Meselson & Stahl (1958)




AP Biology
                                         1908 | 1933
   Genes are on chromosomes
   § T.H. Morgan
        u working with Drosophila
          (fruit flies)
        u genes are on
          chromosomes
        u 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
AP Biology     about proteins?!
                                              1928
   The “Transforming Factor”
    § Frederick Griffith
         u   Streptococcus pneumonia
             bacteria
             § was working to find cure for
               pneumonia
         u harmless live bacteria mixed
           with heat-killed infectious
           bacteria causes disease in mice
         u substance passed from dead
           bacteria to live bacteria =
           “Transforming Factor”

AP Biology
      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




   Transformation?
   something in heat-killed bacteria could still transmit
   disease-causing properties
  AP Biology
                                                        1944
   DNA is the “Transforming Factor”
    § Avery, McCarty & MacLeod
         u   purified both DNA & proteins from
             Streptococcus pneumonia bacteria
              § which will transform non-pathogenic bacteria?
         u   injected protein into bacteria
              § no effect
         u   injected DNA into bacteria         What’s the
              § transformed harmless bacteria   conclusion?

                into virulent bacteria


AP Biology
   Avery, McCarty & MacLeod




    Oswald Avery
                                    Colin MacLeod


AP Biology         Maclyn McCarty
                                                1952 | 1969
   Confirmation of DNA
    § Hershey & Chase
          u classic “blender” experiment
          u worked with bacteriophage

               § viruses that infect bacteria
          u   grew phage viruses in 2 media,
 Why use      radioactively labeled with either
  Sulfur           35
    vs.
               §   S in their proteins
Phosphorus?    § 32P in their DNA
          u   infected bacteria with
              labeled phages
AP Biology
   Hershey & Chase




AP Biology   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            35                           32
                           S radioactivity              P radioactivity found
genetic info?
 AP Biology              found in the medium          in the bacterial cells
AP Biology
   Blender experiment
    § Radioactive phage & bacteria in blender
             35
         u        S phage
              § radioactive proteins stayed in supernatant
              § therefore protein did NOT enter bacteria
             32
         u        P phage
              § radioactive DNA stayed in pellet
              § therefore DNA did enter bacteria
         u   Confirmed DNA is “transforming factor”

                   Taaa-Daaa!

AP Biology
                                                    1947
   Chargaff
    § DNA composition: “Chargaff’s rules”
         u varies from species to species
         u all 4 bases not in equal quantity

         u bases present in characteristic ratio

             § humans:
                         A = 30.9%
                         T = 29.4%    What do
                         G = 19.9%   you notice?!

                         C = 19.8%


AP Biology
                                              1953 | 1962
   Structure of DNA
    § Watson & Crick
         u   developed double helix model of DNA
             § other scientists working on question:
               w Rosalind Franklin
               w Maurice Wilkins
               w Linus Pauling




AP Biology
              Franklin           Wilkins               Pauling
                      1953 article in Nature
   Watson and Crick




AP Biology
   Rosalind Franklin (1920-1958)




AP Biology
  Double helix structure of DNA




 the structure of DNA suggested a mechanism for
 how DNA is copied by the cell
AP Biology
   Directionality of DNA
    § You need to      PO                       nucleotide
                                  4
        number the
        carbons!
         u   it matters!                          N base

                             5¢ CH2
                                            O
                  This
                  will be    4¢                       1¢
               IMPORTANT!!              ribose

                                  3¢             2¢
AP Biology
                                       OH
                                                  5¢
   The DNA backbone                        PO4
    § Putting the DNA
        backbone together                              base
                                            CH2
         u   refer to the 3¢ and 5¢               O
             ends of the DNA                  C
              § the last trailing carbon       O
                                           –
                                             O P O
                                               O          base
                I mean it…                     CH2
                This will be                          O
               IMPORTANT!!


                                                  OH
AP Biology                                         3¢
    Anti-parallel strands
     § Phosphate to sugar bond
         involves carbons in 3¢ & 5¢
         positions
          u DNA molecule has
            “direction”
          u complementary strand
            runs in opposite direction

“It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible copying
mechanism for the genetic material.”
  AP Biology
                           Watson & Crick
  Bonding in DNA
                              hydrogen
                               bonds
                    5’                          3’


   phosphodiester
       bonds


                    3’
                                                5’

….strong or weak bonds?
How do
AP Biology the bonds fit the mechanism for copying DNA?
   Base pairing in DNA
    § Purines
         u adenine (A)
         u guanine (G)

     § Pyrimidines
         u thymine (T)
         u cytosine (C)

     § Pairing
         uA:T
         uC : G


AP Biology
   Copying DNA
    § Replication of DNA
         u   base pairing allows
             each strand to serve
             as a pattern for a
             new strand




AP Biology
                                     verify through
   Models of DNA Replication          experiments…


     § Alternative models
         u   so how is DNA copied?




AP Biology
                                                                1958
   Semi-conservative replication
    § Meselson & Stahl
        u    label nucleotides of “parent” DNA strands with
             heavy nitrogen = 15N
        u    label new nucleotides with lighter isotope = 14N
                           “The Most Beautiful Experiment in Biology”

                          parent    replication
  make predictions…




AP Biology
                                                   1958
   Semi-conservative replication
    § Make predictions…
             15
        u     N strands replicated in 14N medium
        u    1st round of replication?
        u    2nd round?




AP Biology
                                                  let’s meet
   DNA Replication                                the team…


     § Large team of enzymes coordinates replication




AP Biology
   Replication: 1st step
    § Unwind DNA
         u   helicase enzyme
             § unwinds part of DNA helix
             § stabilized by single-stranded binding proteins




    single-stranded binding proteins
AP Biology
             Replication: 2nd step
              § Bring in new nucleotides to
                match up to template strands


                                    But…
                                Where’s the
                                  ENERGY
                               We’re missing
                                  the bonding!
                              forsomething!
                                   What?




AP Biology
   Energy of Replication
     § Where does the energy for the bonding come
        from?

                                         energy
     You
  remember
     ATP!
 Is that the
 only energy
  molecule?




                TTP
                ATP
                CTP
                GTP                     CMP
                                        AMP
                                        ADP
                                        GMP
                                        TMP
AP Biology
   Energy of Replication
     § The nucleotides arrive as nucleosides
         u   DNA bases with P–P–P
         u   DNA bases arrive with their own energy source
             for bonding
         u   bonded by DNA polymerase III




     ATP              GTP            TTP           CTP

AP Biology
                                5'                3'

   Replication                energy
                                       DNA
    § Adding bases                     P III
         u can only add      energy
           nucleotides to 3¢
           end of a growing
           DNA strand        energy
         u strand grow 5'®3’


                              energy
             B.Y.O. ENERGY



                                3'                5'
AP Biology                       leading strand
     5'                   3'            5'                    3'




                          ligase




                               energy
                                        3'
3' Biology
 AP                                     leading strand   5'
      lagging strand 5'
AP Biology
   Leading & Lagging strands
    Leading strand
       - continuous synthesis



                                                Okazaki




                                Lagging strand
                                  - Okazaki fragments
                                  - joined by ligase
AP Biology
                                   - “spot welder” enzyme
   Okazaki fragments




AP Biology
   Priming DNA synthesis
   § DNA polymerase III
      can only extend an
      existing DNA molecule
       u     cannot start new one
             § cannot place first base
       u     short RNA primer is
             built first by primase
             § starter sequences
             § DNA polymerase III can
               now add nucleotides to
               RNA primer



AP Biology
   Cleaning up primers
   DNA polymerase I
   removes sections of
   RNA primer and
   replaces with DNA
   nucleotides




AP Biology
   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
AP Biology
   And in the end…
   § Ends of
       chromosomes
       are eroded with
       each replication
        u an issue in
          aging?
        u ends of
          chromosomes
          are protected by
          telomeres

AP Biology
   Telomeres
   § Expendable,
      non-coding sequences
      at ends of DNA
       u     short sequence of
             bases repeated 1000s
             times
       u     TTAGGG in humans
   § Telomerase enzyme in
      certain cells
       u     enzyme extends
             telomeres
       u     prevalent in cancers
             § Why?


AP Biology
   Replication bubble
  Adds 1000 bases/second!
® Which direction does DNA build?
® List the enzymes & their role




AP Biology
   Replication enzymes
   § helicase
   § DNA polymerase III
   § primase
   § DNA polymerase I
   § ligase
   § single-stranded binding proteins



AP Biology
    DNA polymerases
      § DNA polymerase III
          u   1000 bases/second
          u   main DNA building enzyme
      § DNA polymerase I
          u   20 bases/second
          u   editing, repair & primer removal

DNA polymerase III enzyme




 AP Biology
   Editing & proofreading DNA
     § 1000 bases/second =
        lots of typos!
     § DNA polymerase I
         u   proofreads & corrects
             typos
         u   repairs mismatched bases
         u   excises abnormal bases
             § repairs damage
               throughout life
         u   reduces error rate from
             1 in 10,000 to
             1 in 100 million bases

AP Biology
   Fast & accurate!
    § It takes E. coli <1 hour to copy
        5 million base pairs in its single
        chromosome
         u   divide to form 2 identical daughter cells
     § Human cell copies its 6 billion bases &
        divide into daughter cells in only few
        hours
         u remarkably accurate
         u only ~1 error per 100 million bases

         u ~30 errors per cell cycle
AP Biology
   What’s it really look like?



                                 1




                                         2




                                     3

                            4




AP Biology
   The “Central Dogma”
    § flow of genetic information within a cell

                transcription    translation

   DNA                     RNA            protein


         replication


AP Biology
             Any Questions??




AP Biology

								
To top