Download 5 DNA History Replication

DNA
The Genetic Material
Genetics Lecture #5
AP Biology
2006-2007
Objectives
 Explain how the structure of DNA was
discovered.
 Explain how and why DNA replicates
itself.
AP Biology
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)

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1908 | 1933
Chromosomes related to phenotype
 T.H. Morgan

working with Drosophila
 fruit flies

associated phenotype with
specific chromosome
 white-eyed male had specific
X chromosome
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1908 | 1933
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?
What’s so impressive
about proteins?!
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The “Transforming 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


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“Transforming Principle”
1928
The “Transforming Principle” mix heat-killed
live pathogenic
strain of bacteria
A.
mice die
live non-pathogenic heat-killed
strain of bacteria
pathogenic bacteria
B.
C.
mice live
mice live
pathogenic &
non-pathogenic
bacteria
D.
mice die
Transformation = change in phenotype
something in heat-killed bacteria could still transmit
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disease-causing properties
1944
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
mice die
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What’s the
conclusion?
1944 | ??!!
Avery, McCarty & MacLeod
 Conclusion

first experimental evidence that DNA was the
genetic material
Oswald Avery
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Maclyn McCarty
Colin MacLeod
1952 | 1969
Confirmation of DNA
 Hershey & Chase
classic “blender” experiment
 worked with bacteriophage

 viruses that infect bacteria

Why use
Sulfur
vs.
Phosphorus?

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grew phage viruses in 2 media,
radioactively labeled with either

35S
in their proteins
 32P in their DNA
infected bacteria with
labeled phages
Hershey
Protein coat labeled
with 35S
Hershey
& Chase
DNA labeled with 32P
T2 bacteriophages
are labeled with
radioactive isotopes
S vs. P
bacteriophages infect
bacterial cells
bacterial cells are agitated
to remove viral protein coats
Which
radioactive
marker is found
inside the cell?
Which molecule
carries viral
genetic
info?
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35S
radioactivity
found in the medium
32P
radioactivity found
in the bacterial cells
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Blender experiment
 Radioactive phage & bacteria in blender

35S
phage
 radioactive proteins stayed in supernatant
 therefore viral protein did NOT enter bacteria
 32
P phage
 radioactive DNA stayed in pellet
 therefore viral DNA did enter bacteria

Confirmed DNA is “transforming factor”
Taaa-Daaa!
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1952 | 1969
Hershey
Hershey & Chase
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Martha Chase
Alfred Hershey
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%
AP Biology
That’s interesting!
What do you notice?
Rules
A = T
C = G
1947
1953 | 1962
Structure of DNA
 Watson & Crick

developed double helix model of DNA
 other leading scientists working on question:
 Rosalind Franklin
 Maurice Wilkins
 Linus Pauling
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Franklin
Wilkins
Pauling
Watson and Crick
Watson
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Watson Explains Base pairing
Crick
Rosalind Franklin (1920-1958)
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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
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material.”
— Watson & Crick
Models of DNA Replication
 Alternative models

become experimental predictions
conservative
P
1
2
AP Biology
Can you design
a nifty experiment
to verify?
semiconservative
dispersive
Semiconservative replication
1958
 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”
Make predictions…
15N/15N
15N
parent
strands
AP Biology
parent
replication

Predictions
14N/14N
1st round of
replication
15N/14N
15N/14N
15N/15N
semiconservative
dispersive
conservative
2nd round of
replication

14N/14N
P
15N/15N
1
15N/15N
2 15N parent
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strands

14N/14N
15N/14N

15N/14N
semiconservative
dispersive
conservative
Meselson & Stahl
Matthew Meselson
Franklin Stahl
Franklin Stahl
Matthew Meselson
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

AP Biology
Meselson & Stahl (1958)
 semi-conservative replication
The “Central Dogma”
 Flow of genetic information in a cell
transcription
DNA
replication
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translation
RNA
protein
DNA Replication
AP Biology
2007-2008
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
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material.”
Watson & Crick
Directionality of DNA
 You need to
PO4
nucleotide
number the
carbons!

it matters!
N base
5 CH2
This will be
IMPORTANT!!
O
4
3
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1
ribose
OH
2
The DNA backbone
 Putting the DNA
backbone together

refer to the 3 and 5
ends of the DNA
 the last trailing carbon
Sounds trivial, but…
this will be
IMPORTANT!!
5
PO4
base
5 CH2
O
4
1
C
3
O
–O P O
O
5 CH2
2
base
O
4
1
2
3
OH
AP Biology
3
Anti-parallel strands
 Nucleotides in DNA
backbone are bonded from
phosphate to sugar
between 3 & 5 carbons
5
3
3
5
DNA molecule has
“direction”
 complementary strand runs
in opposite direction

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Bonding in DNA
5
hydrogen
bonds
3
covalent
phosphodiester
bonds
3
5
….strong or weak bonds?
AP
Biology
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
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
Let’s meet
the team…
 Large team of enzymes coordinates replication
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Replication: 1st step
 Unwind DNA

I’d love to be
helicase & unzip
your genes…
helicase enzyme
 unwinds part of DNA helix
 stabilized by single-stranded binding proteins
helicase
single-stranded binding proteins
AP Biology
replication fork
Replication: 2nd step
 Build daughter DNA
strand
add new
complementary bases
 DNA polymerase III

DNA
Polymerase III
AP Biology
But…
Where’s the
We’re missing
ENERGY
something!
for the bonding!
What?
Energy of Replication
Where does energy for bonding usually come from?
We come
with our own
energy!
You
remember
ATP!
Are there
otherenergy
ways
other
to
get energy
nucleotides?
out
it?
You of
bet!
ATP
GTP
CTP
TTP
AP Biology
modified nucleotide
And we
leave behind a
nucleotide!
energy
energy
CMP
TMP
GMP
AMP
ADP
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
AP Biology
GTP
TTP
CTP
5
Replication
 Adding bases

can only add
nucleotides to
3 end of a growing
DNA strand
 need a “starter”
nucleotide to
bond to

strand only grows
53
AP Biology
B.Y.O. ENERGY!
The energy rules
the process
3
energy
DNA
Polymerase III
energy
DNA
Polymerase III
energy
DNA
Polymerase III
DNA
Polymerase III
energy
3
5
Let’s Replicate!!!!
DNA Video
AP Biology