Structure
(chapter 10, pages 266 – 278)
and
Replication of DNA
(chapter 12, pages 318 – 334)
Structure of DNA
• Designate the Nucleotides
– Purines
• Guanine = G
• Adenine = A
– Pyrimidines
• Thymine = T
• Cytosine = C
Structure of DNA
• Nucleotides join together, forming a
polynucleotide chain, by
phosphodiester bonds
– The phosphate attached to the 5’ carbon
on one sugar
– Attaches to the 3’ hydroxyl (OH) group
on the previous nucleotide
5’-phosphate of
last nucleotide
chemically
bonded to the
3’-hydroxyl of
the next-to-last
nucleotide
A phosphodiester bond
Structure of DNA
• DNA is a double helix (two strands)
held together by hydrogen bonds
– Adenine (A) and thymine (T) are paired
– Guanine (G) and cytosine (C) are paired
– Always a purine pairs with a pyrimidine
5’-end
3’-end
(free 3’-OH)
The two polynucleotide
strands (the
backbones) in the
double helix run in
opposite directions,
and are said to be antiparallel
3’-end
5’-end
(free 5’phosphate)
5’-end
3’-end
(free 3’-OH)
Because of the pairing
(A-T; G-C), one
polynucleotide chain is
always complementary
to the base sequence of
the other strand
3’-end
5’-end
(free 5’phosphate)
It has not escaped our notice that
the specific pairing we have
postulated immediately suggests a
possible copying mechanism for the
genetic material.
J. D. Watson and F. H. C. Crick, 1953
Matthew Meselson and Franklin Stahl, 1958
entirely new
AND entirely
old DNA
molecules
present
ALL DNA
molecules
are made up
of both old
and new DNA
entirely new
DNA molecules
present BUT
not entirely old
DNA molecules
Meselson and Stahl
• Experiment
– Grew E. coli in a growth medium
containing only 15N (heavy nitrogen)
(note: Normal isotope is 14N lighter nitrogen)
– Did this for many generations so that all
of the bacterial DNA would be “heavy”
Meselson and Stahl
• Experiment
– Then grew the bacteria with 15N
incorporated in their DNA in medium
containing only 14N (would be
incorporated into the new DNA)
This way they could differentiate the
original DNA from newly incorporated
DNA
Meselson and Stahl
• Experiment
– At each generation
• Isolated the DNA
• Looked at the density of the DNA in a CsCl
gradient
Matthew Meselson and Franklin Stahl, 1958
What Meselson and Stahl expected if
semiconservative replication
entirely new
DNA molecules
present BUT
not entirely old
DNA molecules
First generation
results helped
them rule out
one of the three
possible mode
of replication
Matthew Meselson and Franklin Stahl, 1958
entirely new
AND entirely
old DNA
molecules
present
ALL DNA
molecules
are made up
of both old
and new DNA
entirely new
DNA molecules
present BUT
not entirely old
DNA molecules
Clincher evidence!
Why?
In dispersive model
lighter DNA band
should not have
formed
Great test question:
Predict what the
cesium chloride
gradients would look
like for conservative
and dispersive
replication!
Should be able to draw something like this for
conservative and dispersive
Meselson and
Stahl showed that
the
semiconservative
pattern of
replication is what
was found
So the DNA double
helix unwinds and
each strand acts as a
template for
replication of the new
half
Replication
General features:
1. There is a specific site where replication begins (origin)
which must be recognized
2. The two strands of DNA must be separated
3. The original strand becomes the template for the new
DNA strand
4. A primer molecule must be added on which the new
DNA chain can be built
5. New nucleotides must be added complementary to the
template strand
6. The newly synthesized DNA must be edited and joined
into one continuous molecule
Replication
Origin of replication
– Where synthesis of new DNA begins
– A specific location with a specific sequence of
nucleotides
In some organisms a specific
location that can be mapped
Multiple and random origins in eukaryotes
Origins of replication
In bacteria and viruses
one origin of replication
In eukaryotes there can
be thousands of
replication origins
Initiator Proteins
Start to denature
the DNA so each
strand can act as
a template
Recognizes
the Origin
of Replication
Replication
• DNA is unwound by a helicase
– Separates the double helix by breaking
the hydrogen bonds
Helicase
The separated (single strand DNA) is
combined with single-strand binding proteins
•Protects DNA from degradation
•Keeps the complementary strands from rejoining
Replication
• As DNA is unwound it will tangle and
knot, called supercoiling (from the
unwinding of the helix)
The supercoiling must be relaxed (the DNA
unknotted)
This is done by a class of enzymes called
topisomerases (gyrase)
2
3
1
1 = initiator proteins
2 = single strand binding proteins
3 = helicase
4 = topoisomerase (gyrase)
4
Replication
DNA polymerases
– Enzymes that synthesize new DNA
5’ triphosphates
of the four
nucleotides
must be present
(dATP, dGTP,
dTTP, dCTP)
Two of the
phosphates
are cleavedoff, providing
energy to run
the reaction
The preexisting single
strand of DNA is the
template strand
Phosphates
cleaved to
provide energy
for the reaction
Nucleotide
monophosphates
are then joined to
the 3’OH group
Complementary base to the template strand
Replication
• DNA Polymerase
– Must have a free 3’-OH group onto
which to add the new nucleotides
– No known DNA polymerase is able to
initiate chains; thus, requires a primer
to start synthesis
DNA is always polymerized in a 5’ to 3’ direction
and antiparallel to the template strand
KNOW ALL TERMS!
Replication
• DNA Polymerase
– Must have a free 3’-OH group onto
which to add the new nucleotides
– No known DNA polymerase is able to
initiate chains; thus, requires a primer
to start synthesis
Must have a primer
(which is an RNA
molecule)
The primer is
synthesized by the
enzyme primase (RNA
polymerase)
Replication
• Two DNA polymerase enzymes are
necessary for replication in E. coli
– DNA polymerase I
– DNA polymerase III
• Both have polymerase and
exonuclease activities (functions)
Replication
• Polymerase:
– Synthesize new DNA in the 5’  3’ direction
• Exonuclease:
– Remove nucleotides from the end of a chain
(proofreading and editing functions)
• 5’  3’ (removes primers)
• 3’  5’ (editing, removes incorrect bases)
5’ 3’
exonuclease
activity
Replication
• DNA Polymerase I
– Synthesize new DNA in the 5’  3’ direction
• Only synthesizes short sequences of new DNA
– 3’  5’ exonuclease activity (proofreading)
– 5’  3’ exonuclease activity (remove primers)
• DNA Polymerase III
– Synthesize new DNA in the 5’  3’ direction
• Synthesizes long sequences of new DNA
– 3’  5’ exonuclease activity (proofreading)
Replication
• The phosphodiester backbone of
DNA must be joined
• This is done by the enzyme ligase
The phosphodiester
backbone of DNA
must be joined