dna the genetic material · semiconservative replication meselson & stahl label “parent”...

Click here to load reader

Post on 24-May-2020

4 views

Category:

Documents

1 download

Embed Size (px)

TRANSCRIPT

  • AP Biology 2006-2007

    DNA

    The Genetic Material

  • AP Biology

    Chromosomes related to phenotype

    T.H. Morgan

    working with Drosophila

    fruit flies

    associated phenotype with

    specific chromosome

    white-eyed male had specific

    X chromosome

    1908 | 1933

  • AP Biology

    Genes are on chromosomes

    Morgan’s conclusions

    genes are on chromosomes

    but is it the protein or the

    DNA of the chromosomes

    that are the genes?

    initially proteins were thought

    to be genetic material…

    Why?

    1908 | 1933

  • AP Biology

    The “Transforming Principle” 1928

    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”

  • AP Biology

    The “Transforming Principle”

    Transformation = change in phenotype

    something in heat-killed bacteria could still transmit

    disease-causing properties

    live pathogenicstrain of bacteria

    live non-pathogenicstrain of bacteria

    mice die mice live

    heat-killed pathogenic bacteria

    mix heat-killed pathogenic & non-pathogenicbacteria

    mice live mice die

    A. B. C. D.

  • AP Biology

    DNA is the “Transforming 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

    1944

    mice die

  • AP BiologyOswald Avery Maclyn McCarty Colin MacLeod

    Avery, McCarty & MacLeod Conclusion

    first experimental evidence that DNA was the genetic material

    1944 | ??!!

  • AP Biology

    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

    1952 | 1969Hershey

  • AP Biology

    Protein coat labeledwith 35S

    DNA labeled with 32P

    bacteriophages infectbacterial cells

    T2 bacteriophagesare labeled with

    radioactive isotopesS vs. P

    bacterial cells are agitatedto remove viral protein coats

    35S radioactivityfound in the medium

    32P radioactivity foundin the bacterial cells

    Which radioactive marker is found inside the cell?

    Which molecule carries viral genetic info?

    Hershey

    & Chase

  • AP Biology

    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”

  • AP Biology

    Hershey & Chase

    Alfred HersheyMartha Chase

    1952 | 1969Hershey

  • AP Biology

    Chargaff

    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%

    1947

    RulesA = TC = G

  • AP Biology

    Rosalind Franklin (1920-1958)

  • AP Biology

    Structure of DNA

    Watson & Crick

    developed double helix model of DNA

    other leading scientists working on question:

    Rosalind Franklin

    Maurice Wilkins

    Linus Pauling

    1953 | 1962

    Franklin Wilkins Pauling

  • AP Biology

    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

  • AP Biology

    Watson and Crick1953 article in Nature

    CrickWatson

    file://localhost/Users/kfoglia/Desktop/watsoncrick.pdf

  • AP Biology

    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

  • AP Biology

    Models of DNA Replication Alternative models

    become experimental predictions

    conservative semiconservative dispersive

    1

    2

    P

  • AP Biology

    Semiconservative replication Meselson & Stahl

    label “parent” nucleotides in DNA strands with heavy nitrogen = 15N

    label new nucleotides with lighter isotope = 14N

    “The Most Beautiful Experiment in Biology”

    1958

    parent replication

    15N parent

    strands

    15N/15N

  • AP Biology

    Predictions

    1st round of

    replication

    conservative

    15N/15N

    14N/14N

    semi-

    conservative

    15N/14N

    dispersive

    15N/14N

    conservative

    15N/15N

    14N/14N

    semi-

    conservative

    15N/14N

    dispersive

    15N/14N

    2nd round of

    replication

    14N/14N

    15N parent

    strands

    15N/15N

    12

    P

  • AP Biology

    Franklin Stahl

    Matthew Meselson

    Matthew Meselson Franklin Stahl

    Meselson & Stahl

  • AP Biology

    Scientific History March to understanding that DNA is the genetic material

    T.H. Morgan (1908)

    genes are on chromosomes

    Frederick Griffith (1928)

    a transforming factor can change phenotype

    Avery, McCarty & MacLeod (1944)

    transforming factor is DNA

    Erwin Chargaff (1947)

    Chargaff rules: A = T, C = G

    Hershey & Chase (1952)

    confirmation that DNA is genetic material

    Watson & Crick (1953)

    determined double helix structure of DNA

    Meselson & Stahl (1958)

    semi-conservative replication

  • AP Biology 2007-2008

    DNA Replication

  • AP Biology

    The DNA backbone

    Putting the DNA

    backbone together

    refer to the 3 and 5

    ends of the DNA

    the last trailing carbon

    OH

    O

    3

    PO4

    base

    CH2O

    base

    O

    P

    O

    C

    O–O

    CH2

    1

    2

    4

    5

    1

    2

    3

    3

    4

    5

    5

  • AP Biology

    Anti-parallel strands

    Nucleotides in DNA

    backbone are bonded from

    phosphate to sugar

    between 3 & 5 carbons

    DNA molecule has

    “direction”

    complementary strand runs

    in opposite direction

    3

    5

    5

    3

  • AP Biology

    Bonding in DNA

    ….strong or weak bonds?

    How do the bonds fit the mechanism for copying DNA?

    3

    5 3

    5

    covalent

    bonds

    hydrogen

    bonds

  • AP Biology

    Base pairing in DNA

    Purines

    adenine (A)

    guanine (G)

    Pyrimidines

    thymine (T)

    cytosine (C)

    Pairing

    A : T 2 bonds

    C : G 3 bonds

  • AP Biology

    Copying DNA

    Replication of DNA

    base pairing allows each strand to serve as a template for a new strand

    new strand is 1/2 parent template & 1/2 new DNA semi-conservative

    copy process

  • AP Biology

    DNA Replication Large team of enzymes coordinates replication

  • AP Biology

    Replication: 1st step

    Unwind DNA

    helicase enzyme

    unwinds part of DNA helix

    stabilized by single-stranded binding proteins

    single-stranded binding proteinsreplication fork

    helicase

  • AP Biology

    DNA

    Polymerase III

    Replication: 2nd step

    But…We’re missing

    something!What?

    Where’s theENERGY

    for the bonding!

    Build daughter DNA

    strand

    add new

    complementary bases

    DNA polymerase III

  • AP Biology

    Energy of Replication The nucleotides arrive as nucleosides

    DNA bases with P–P–P P-P-P = energy for bonding

    DNA bases arrive with their own energy source for bonding

    bonded by enzyme: DNA polymerase III

    ATP GTP TTP CTP

  • AP Biology

    Adding bases

    can only add

    nucleotides to

    3 end of a growing

    DNA strand

    need a “starter”

    nucleotide to

    bond to

    strand only grows

    53

    DNA

    Polymerase III

    DNA

    Polymerase III

    DNA

    Polymerase III

    DNA

    Polymerase III

    energy

    energy

    energy

    Replication energy

    3

    3

    5

    5

  • AP Biology

    Limits of DNA polymerase III

    can only build onto 3 end of

    an existing DNA strand

    Leading & Lagging strands

    5

    5

    5

    5

    3

    3

    3

    5

    35

    3 3

    Leading strand

    Lagging strandligase

    Okazaki

    Leading strand

    continuous synthesis

    Lagging strand

    Okazaki fragments

    joined by ligase

    “spot welder” enzyme

    DNA polymerase III

    3

    5

    growing replication fork

  • AP Biology

    DNA polymerase III

    Replication fork / Replication bubble

    5

    35

    3

    leading strand

    lagging strand

    leading strand

    lagging strandleading strand

    5

    3

    3

    5

    5

    3

    5

    3

    5

    3 5

    3

    growing replication fork

    growing replication fork

    5

    5

    5

    5

    5

    3

    3

    5

    5lagging strand

    5 3

  • AP Biology

    DNA polymerase III

    RNA primer

    built by primase

    serves as starter sequence for DNA polymerase III

    Limits of DNA polymerase III

    can only build onto 3 end of

    an existing DNA strand

    Starting DNA synthesis: RNA primers

    5

    5

    5

    3

    3

    3

    5

    35

    3 5 3

    growing replication fork

    primase

    RNA

  • AP Biology

    DNA polymerase I

    removes sections of RNA

    primer and replaces with

    DNA nucleotides

    But DNA polymerase I still

    can only build onto 3 end of

    an existing DNA strand

    Replacing RNA primers with DNA

    5

    5

    5

    5

    3

    3

    3

    3

    growing replication fork

    DNA polymerase I

    RNA

    ligase

  • AP Biology

    Replication fork

    3’

    5’

    3’

    5’

    5’

    3’

    3’ 5’

    helicase

    direction of replication

    SSB = single-stranded binding proteins

    primase

    DNA polymerase III

    DNA polymerase III

    DNA polymerase I

    ligase

    Okazaki fragments

    leading strand

    lagging strand

    SSB

  • AP Biology

    DNA polymerases

    DNA polymerase III

    1000 bases/second!

    main DNA builder

    DNA polymerase I

    20 bases/second

    editing, repair & primer removal

    DNA polymerase III enzyme

    Arthur Kornberg1959

    Roger Kornberg2006

  • AP Biology

    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

  • AP Biology

    Loss of bases at 5 ends

    in every replication

    chromosomes get shorter with each replication

    limit to number of cell divisions?

    DNA polymerase III

    All DNA polymerases can

    only add to 3 end of an

    existing DNA strand

    Chromosome erosion

    5

    5

    5

    5

    3

    3

    3

    3

    growing replication fork

    DNA polymerase I

    RNA

  • AP Biology

    Repeating, non-coding sequences at the end

    of chromosomes = protective cap

    limit to ~50 cell divisions

    Telomerase

    enzyme extends telomeres

    can add DNA bases at 5 end

    different level of activity in different cells

    high in stem cells & cancers -- Why?

    telomerase

    Telomeres

    5

    5

    5

    5

    3

    3

    3

    3

    growing replication fork

    TTAAGGGTTAAGGG