Whiteboard Review
Draw what Meselson and Stahl would have
observed in the centrifuge tube after cycle 1 if
DNA replication was
● conservative.
● semiconservative.
● dispersive.
Whiteboard Review
Start with “heavy” DNA.
Draw the DNA strands after 4 cycles of
replication with “light” DNA.
DNA Replication
“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 (1953)
Whiteboard
Draw a DNA nucleotide.
Hint:
circle
pentagon
rectangle
My image doesn’t have a rectangle as a base.
Oh well.
Whiteboard
Draw a second DNA
nucleotide covalently
connected to your previous
drawing.
Whiteboard
Draw a two additional DNA
nucleotides hydrogen bonded
to your previous drawing.
Be Prepared to Answer this Question
How do the bonds found in
DNA fit the mechanism for
copying DNA?
BILL:
Watch and listen to the animation. Write down
all key terms related to DNA replication.
How nucleotides are added in DNA
replication
1. Helicase
breaks hydrogen
bonds between
bases, unzips and
unwinds the helix
DANCE MOVE:
Helicase breaks hydrogen bonds between complementary strands of parent DNA molecule
Counts 1 and 2
Hands together move straight out from body.
Represents the replication fork and the action
of helicase creating two single strands at the
replication fork.
Whiteboard
Do not erase when done...
● Draw a replication BUBBLE of DNA.
● Use an arrow to show where the origin of
replication is located
● Label the 5’ and 3’ end of each DNA strand
(you can decide which is which, just be sure it’s antiparallel)
● Add two helicase enzymes, one at each
FORK.
Usually drawn as ovals
2. Gyrase
AKA “topoisomerase”
Ahead of the replication fork, gyrase
unwinds the DNA supercoil
Whiteboard
Add a gyrase to each replication fork.
3. Single Stranded Binding Proteins
Hold the DNA
strands apart
(keeps the
separated
strands apart and
stabilize the
unwound DNA).
DANCE MOVE:
Single stranded binding proteins prevent the parent DNA molecules from reconnecting
Counts 3 and 4
Arms move towards each other and away from
each other two times. Represents the DNA
strands “wanting” to reconnect, but not being
able to.
Whiteboard
Add SSBP to each replication fork.
4. PRIMASE AND PRIMERS
“Primer”
DANCE MOVE:
Primase adds an RNA primer onto the leading strand
Count 5
Right hand with 5 fingers moves to left
shoulder. The right hand represents primase
placing the primer.
Whiteboard
Create a table comparing DNA to RNA
5. DNA Polymerase III
DNA polymerase III
adds DNA nucleoside
triphosphates to the Huh?
RNA primer sequence
in a 5’ to 3’ direction. What?
Ponder…..
Where does energy for building DNA
come from?
Whiteboard
Draw ATP, GTP, TTP, and CTP
Hint:
circles
pentagon
rectangles of different size
Adding Bases
DNA polymerase III can only add nucleotides to
3’ end of a growing DNA strand
–needs a “starter” nucleotide to bond to
New strand only grows 5’ → 3’
Whiteboard
Draw this…
Label additional 5’ and 3’ ends
3’
Whiteboard
In a different color, add the new DNA strands
that are being made and label their 3’ and 5’
ends.
3’
Whiteboard
What is the “problem” or “limitation” with DNA
polymerase III only being able to add to the 3’ end
of a growing strand?
Add in DNA polymerase III to show it building
new DNA at the 3’ end of the new strands.
3’
Leading Strand
DNA polymerase III
can synthesize a
complementary
strand on one side of
the template in the 5’
to 3’ direction with no
problem
DANCE MOVE:
DNA polymerase III builds off the primer and moves in the same direction as helicase, towards the
replication fork, building a complementary strand. It begins at the 5’ end of the new daughter strand
and moves in the 3’ direction.
Counts 6 and 7
Right hand on left shoulder starts with 5 fingers
out (from the previous step). This represents
the 5’ end of the daughter strand. Then the
hand flips to two fingers out and to 3 fingers
out. The fingers change from the 5 to 2 to 3
representing DNA polymerase III moving from
the 5’ to 3’ end of the daugher strand (5’ “to” 3’)
DANCE MOVE:
DNA polymerase III moves in a continuous motion down the leading strand of the DNA template.
Count 8
The right hand with 3 fingers moves along the left
hand towards the wrist. At the end of the motion,
your two hands should be next to each other. The
left hand has 5 fingers exposed (representing the
5’ end of the parent DNA strand) and the right
hand has 3 fingers expose (representing the 3’ end
of the daughter DNA strand). Look! Your hands
are antiparallel!
What about the other strand??
Lagging Strand
DNA polymerase III must work
away from the replication fork.
Makes a short strand of DNA,
called an Okazaki fragment.
As the bubble widens, it can
make another short strand,
and so on.
DANCE MOVE:
Now we’ll replicate the lagging strand. Again, the primase adds an RNA primer
Count 1
The left hand with 5 fingers moves towards the
elbow of the right arm
DANCE MOVE:
DNA polymerase III also works in a 5’ to 3’ direction on the growing lagging strand.
Counts 2 and 3
The left hand on the right elbow still has 5
fingers out (from the previous step). This
represents the 5’ end of the daughter strand.
Then the hand flips to two fingers out and then
to 3 fingers out. The fingers change from the 5
to 2 to 3 representing DNA polymerase III
moving from the 5’ to the 3’ end of the daughter
strand.
DANCE MOVE:
DNA polymerase III adds complementary DNA nucleotides to the lagging strand working away from
the replication fork creating a short Okazaki fragment.
Count 4
The left hand, still with three fingers out, moves
towards the right shoulder (in the opposite
direction as the replication fork was opening).
DANCE MOVE:
To create another Okazaki fragment, again the primase adds an RNA primer.
Count 5
The left hand with five fingers moves towards
the wrist of the right arm.
DANCE MOVE:
DNA polymerase III still works in a 5’ to 3’ direction on the growing lagging strand.
Counts 6 and 7
The left hand on the right wrist still has 5
fingers out (from the previous step). This
represents the 5’ end of the daughter strand.
Then the hand flips to two fingers out and then
to 3 fingers out. The fingers change from the 5
to 2 to 3 representing DNA polymerase III
moving from the 5’ to the 3’ end of the daughter
strand.
DANCE MOVE:
DNA polymerase III adds complementary DNA nucleotides to the lagging strand working away from
the replication fork, creating a second short Okazaki fragment.
Count 4
The left hand, still with three fingers out, moves
towards the right elbow (in the opposite
direction as the replication fork was opening).
6. DNA Polymerase I
RNA primers are
removed and
replaced with
DNA nucleotides
by DNA
Polymerase I.
DANCE MOVE:
DNA polymerase I removes the RNA primers from the leading strand (only 1).
Counts 1 and 2
Fingers on right hand double “brush off the left
shoulder (the spot where the primer was
added)
DANCE MOVE:
DNA polymerase I removes the RNA primers from the leading strand (only 1).
Counts 3, 4, 5, and 6
Fingers on left hand double “brush off the right
elbow and then the right wrist (the sports where
the primer as added)
7. DNA Ligase
Along the lagging
strand the Okazaki
fragments are joined
by DNA Ligase to
form a single DNA
strand.
DANCE MOVE:
Ligase seals up the breaks in the sugar phosphate backbone between the Okazaki fragments.
Counts 7 and 8 and 1 and 2
Left hand circles around the right wrist, as if
“taping” the wrist with a sports tape wrap.
8. Proofreading
DNA polymerase I
–proofreads & corrects typos
–repairs mismatched bases
–removes abnormal bases
–reduces error rate from
1 in 10,000 to
1 in 100 million bases
DANCE MOVE:
Enzymes double check to make sure the replication preceded properly with little mutation.
Counts 3 to 8
As if holding a book up to the nose and reading
across the page, move your head from left to
right between hands in front of your face.
Imagine you are “proofreading.”
How nucleotides are added in DNA
replication
7.1.U2: reference covalent and hydrogen bonds and how
the ease (or not) of breaking the bonds relates to DNA
replication
7.1.U5: create a table with enzyme name and function
2.7.U2: draw a picture showing the location and function
helicase
2.7.U3: explain how DNA poly III uses a template
7.1.U3: draw a picture showing DNA polymerase adding a
nucleotide (circle, pentagon, rectangle) to a primer in a 5’ to
3’ direction
7.1.U4: compare replication on the leading and lagging
strand