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DNA
AND ITS ROLE IN HEREDITY
2006-2007
DNA is the genetic material:
a short history
- DNA was found in the nucleus by Miescher (1868)
• Early in the 20th century, the search for genetic material
led to DNA
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T. H. Morgan’s group (1908): genes are on chromosomes
Frederick Griffith (1928): experiments on S. pneumoniae
Oswald Avery (1944): confirmed Griffith’s experiments
Hershey and Chase (1952): DNA is the genetic material
Erwin Chargaff (1947): The amount of thymine = adenine
Watson and Crick (1953): Structure of DNA
Rosalind Franklin (1951): X-Ray Structure of DNA
Meselson and Stahl (1958): DNA Replication
DNA was found in chromosomes using
dyes that bind specifically to DNA.
Avery, McCarty & MacLeod
injected protein into bacteria
- no effect
injected DNA into bacteria
- transformed harmless bacteria into virulent bacteria
• Conclusion
– first experimental evidence that DNA was the genetic
material
Oswald Avery
Maclyn McCarty
Colin MacLeod
Evidence That Viral DNA Can Program Cells
• Bacteriophages (or phages), are viruses that infect
bacteria
T2 Phage
Protein coat labeled
with 35S
T2 bacteriophages
are labeled with
radioactive isotopes
S vs. P
Hershey
& Chase
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?
DNA labeled with 32P
35S
radioactivity
found in the medium
32P
radioactivity found
in the bacterial cells
DNA Structure Reflects Its Role as the
Genetic Material
• After identifying DNA as the genetic
material, scientists hoped to answer two
questions about the structure:
1. How is DNA replicated between cell
divisions?
2. How does it direct the synthesis of
specific proteins?
Structure of DNA
• How does the structure of DNA account for its role
in genetic inheritance?
• Maurice Wilkins and Rosalind Franklin – used X-ray
crystallography to study molecular structure
Sugar–phosphate
backbone
5 end
Nitrogenous
bases
DNA STRUCTURE
Thymine (T)
Adenine (A)
Cytosine (C)
Chargaff’s rules state that in any species there
is an equal number of A and T bases, and an
equal number of G and C bases
DNA nucleotide
Phosphate
Sugar (deoxyribose)
3 end
Erwin Chargaff reported (1947) that DNA
composition varies from one species to the
next.
Guanine (G)
Fig. 16-5
Watson and Crick
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
5 end
Watson and Crick reasoned that the pairing was specific, dictated
by the base structures
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data
DNA in the Nucleus and in the Cell Cycle
DNA Is a Double
Helix
Base Pairs in
DNA Can
Interact with
Other
Molecules
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
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
Models of DNA Replication
• Alternative models
– become experimental predictions
conservative
P
1
2
semiconservative
dispersive
DNA Replication
• Semiconservative replication
• Each half of the double helix
acquires a new mate
• Each new DNA molecule,
then, is really half old and
half new
5
DNA has directionality
• Putting the DNA backbone
together
– refer to the 3 and 5 ends
of the DNA
• the last trailing carbon
PO4
base
5 CH2
O
4
1
C
3
O
–O P O
O
5 CH2
2
base
O
4
1
2
3
OH
3
Each New DNA Strand Grows by the Addition of
Nucleotides to Its 3′ End
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
Fig. 16-13
Replication: 1st step
Primase
Single-strand binding
proteins
3
Topoisomerase
5
3
5
Helicase
5
RNA
primer
3
Fig. 16-13
Replication: 2nd step
Primase
Single-strand binding
proteins
3
Topoisomerase
5
RNA
primer
5
3
5
Helicase
Primase starts an RNA chain from scratch
- adds RNA nucleotides one at a time using the parental DNA as a template
- 3 end serves as the starting point for the new DNA strand
3
Replication: 2nd step
 Build daughter DNA strand
add new complementary
bases to the 3’ end of the
RNA primer
 DNA polymerase III

DNA
Polymerase III
What is driving polymerization?
New
strand 5
end
Sugar
Phosphate
Template
strand 3 end
5 end
3
end
T
A
T
C
G
C
G
G
C
G
C
T
A
A
Base
3 end
DNA
polymerase
A
3 end
C
Nucleoside
triphosphate
5 end
C
5 end
Fig. 16-16a
Overview
Origin of replication
Leading strand
Lagging strand
Lagging strand
2
1
Leading strand
Overall directions
of replication
DNA Replication Animation
• To summarize, at the replication
fork, the leading stand is copied
continuously into the fork from a
single primer.
• The lagging
strand is
copied away
from the fork
in short
segments, each
requiring a
new primer.
Fig. 16.16
3
Template
strand
5
5
The Lagging Strand: A Closer Look
3
3
Template
strand
3
5
5
RNA primer
3
1
3
5
5
DNA Pol III works in
the direction away
from the replication
fork
3
Template
strand
3
Okazaki
5
5
RNA primer
3
3
1
5
5
Okazaki
fragment
3
1
5
3
5
3
Okazaki
5
5
Template
strand
3
RNA primer
3
3
1
5
5
3
5
Okazaki
fragment
3
1
5
3
5
2
1
3
5
3
Okazaki
5
5
Template
strand
3
RNA primer
3
3
1
5
5
3
1
5
3
5
2
3
3
5
Okazaki
fragment
1
3
5
5
3
5
2
1
3
Okazaki
5
5
Template
strand
3
RNA primer
3
3
1
5
5
3
1
5
3
5
2
3
3
5
Okazaki
fragment
3
5
1
5
3
5
2
1
5
3
1
2
Overall direction of replication
3
5
Chromosome erosion
All DNA polymerases can only
add to 3 end of an existing
DNA strand
DNA polymerase I
5
3
3
5
5
growing
replication fork
3
DNA polymerase III
RNA
Loss of bases at 5 ends
in every replication


chromosomes get shorter with each replication
limit to number of cell divisions?
5
3
Telomeres
Repeating, non-coding sequences at the end of
chromosomes = protective cap

limit to ~50 cell divisions
5
3
3
5
growing
replication fork
5
3
telomerase
5
Telomerase



enzyme extends telomeres
can add DNA bases at 5 end
different level of activity in different cells
 high in stem cells & cancers -- Why?
TTAAGGG TTAAGGG 3
Fig. 16-20
1 µm
Correcting Mistakes
more than 130 DNA repair enzymes have been identified in
humans
• An enzyme detects something wrong in one
strand of the DNA and removes it
• Then DNA polymerase copies the information
in the intact second strand and creates a new
stretch of DNA
• DNA ligase seals the gap
Nobel Prize in Chemistry 2015 Interview
Sunburn Damages DNA
Nucleotide excision repair
Nuclease – a DNA cutting
enzyme
• In mismatch repair, repair
enzymes fix incorrectly paired
nucleotides.
– A hereditary defect in
one of these enzymes
is associated with a
form of colon cancer.
Ghosts of Lectures Past
Frederick Griffith
The “Transforming Principle”
live pathogenic
strain of bacteria
A.
mice die
live non-pathogenic
strain of bacteria
B.
mice live
heat-killed
pathogenic bacteria
C.
mice live
mix heat-killed
pathogenic &
non-pathogenic
bacteria
D.
mice die
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”
parent
15N/15N
15N
parent
strands
replication
1958
P
1
2
conservative
P
1
2
semiconservative
dispersive
DNA: Count the Carbons!
3
’
5’
3’
5’
3’
Okazaki
Leading & Lagging strands
Limits of DNA polymerase III

can only build onto 3 end of an existing DNA
strand
5
3
5
3
5
growing
replication fork
3
5
5
5
Lagging strand
ligase
3
Leading strand
3
Lagging strand



3
Okazaki fragments
joined by ligase
 “spot welder” enzyme

5
3
DNA polymerase III
Leading strand

continuous synthesis
Replacing RNA primers with DNA
DNA polymerase I

removes sections of RNA primer and
replaces with DNA nucleotides
DNA polymerase I
5
3
3
5
5
growing
replication fork
ligase
3
RNA
5
3
But DNA polymerase I still can
only build onto 3 end of an
existing DNA strand