Broad Course Objectives for DNA Replication
Students will be able to:
• describe the historic experiment that demonstrated
DNA replication follows a semi-conservative model.
• describe the process of DNA replication in
prokaryotes at the biochemical level
• explain how proofreading and repair is accomplished
during DNA synthesis
Outline/study guide—DNA Replication
•
At what point in the cell cycle does DNA replication occur?
•
When two DNA molecules (or chromosomes) are made from one, where
do the parental strands end up, vs. the newly synthesized strands? (i.e.
semiconservative replication)
Why can DNA only be synthesized in the 5’  3’ direction?
•
•
•
•
•
What are the enzymes and proteins involved in DNA synthesis? What is
the function of each and at what point do they act?
At what point does RNA function in DNA replication?
What determines the lagging strand vs. the leading strand? How does
this change on the “other” side of the replication origin?
How are the Okazaki fragments joined into one continuous DNA strand?
•
How does the DNA replication machinery correct errors made during
replication?
•
•
Are human chromosomes linear or circular? Bacteria?
Why do linear chromosomes (but not circular chromosomes) have a
problem with telomeres becoming shorter and shorter with each round of
replication? How do some cells get around this?
48 year old woman with Werner Syndrome
Progeria
Each strand of
the parent DNA
molecule
becomes a
template for the
new
molecule(s)
5′
3′
5′
C G
A T
C G
A T
C G
T A
G C
GC
T A
A T
C G
A T
G C
GC
T A
C G
A T
C G
C G
Replication
fork
A T
A
T
C
3′
G
A
G
5′
C
T A
Identical
base sequences
Lagging
A T
strand
5′
3′
T A
T A
C G
C G
C G
A T
Incoming
C G
T A
T A
nucleotides
T A
Leading
G C
strand
C
GC
G C
T A
G
C G
A T
A
5′
3′ T A
C G
A T
Original
Newly
Original
G C
(template) synthesized
(template)
G
C
strand
daughter strand
strand
T A
3′
5′
A T
(a) The mechanism of DNA replication
Brooker Fig 13.1
The width of the
nucleotides
reflect larger
purines and
smaller
pyrimidines
T
C
G
T A
3′
3′
3′
C G
A T
C G
T A
G C
G C
T A
A T
C G
A T
G C
G C
T A
3′
5′
(b) The products of replication
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Different models of DNA Replication
Original
DNA
First
replication
Second
replication
Conservative model
Semiconservative model
Dispersive model
Brooker fig 13.2
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How do you expect the different models to appear in the
centrifuge experiment?
Original
DNA (N15)
First
Replication
(N14)
Second
Replication
(N14)
Conservative model
Semiconservative model
Dispersive model
Brooker fig 13.2
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Experiment to distinguish between DNA replication models
Experimental level
1. Grow bacteria in excess of 15N-containing
compounds. Switch to 14N at Generation 1.
15N-DNA = purple
14N-DNA = blue
Conceptual level
14N
Generation
0
solution
Add
14N
Suspension of
bacterial
cells labeled
with 15N
1
2. Incubate the cells for various cell generations
2
3. Lyse cells to release DNA
37°C
Up to 4 generations
DNA
Cell wall
Lysate
4. Load sample of lysate onto CsCl gradient.
5. Centrifuge until the DNA molecules reach equilibrium
densities.
CsCl
gradient
Cell membrane
Density centrifugation
Light DNA
Half-heavy DNA
6. View DNA within the gradient using a UV light.
Brooker fig 13.3
Heavy DNA
(after 2 generations.)
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Interpreting the Data
Generations After
4.1
3.0
2.5
1.9
1.5
1.1
14N
Addition
1.0
0.7
0.3
Light
Half-heavy
Heavy
*Data from: Meselson, M. and Stahl, F.W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671−682
After ~ two generations: DNA
is “light” and “half-heavy”
(Consistent with which model?)
After one generation,
DNA is “half-heavy”
(consistent with both semiconservative and dispersive
models)
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Why does DNA (and RNA) only “grow” in the 3’ direction?
New strand
Template strand
Sugar
A
Base
Phosphate
3’ end
5’ end
3’ end
5’ end
T
A
T
C
G
C
G
G
C
G
C
A
T
A
P
OH
P
Pyrophosphate 3’ end
C
C
OH
Nucleoside
triphosphate
(Like Brooker, fig 13-15)
2 P
5’ end
5’ end
Fig from Cambell and Reece, 7th ed
Overview of
DNA
Replication
Origin of replication
DNA strands separate at
origin
Primers initiate DNA synthesis.
Synthesis of the leading strand occurs in
the same direction as movement of the
replication fork. 1st Okazaki
fragment of lagging strand is
made in opposite direction.
Replication
forks
Leading
strand
5′
3′
5′
3′
3′
5′
Direction of
replication fork
Primer
The leading strand elongates,
and a second Okazaki fragment
is made.
Primer
3′
5′
1st Okazaki fragment
of the lagging strand
3′
5′
5′
3′
3′
2nd Okazaki
fragment
1st Okazaki
fragment
5′
3′
5′
The leading strand continues to elongate. A
third Okazaki fragment is made, and the first
and second are connected together.
3′
5′
3′
5′
3rd
Okazaki
fragment
3′
5′
Brooker, fig 13.10
First and second Okazaki
fragments have been
connected to each other.
3′
5′
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Functions of key proteins involved with DNA
replication
• DNA helicase breaks the hydrogen
bonds between the DNA strands.
• Topoisomerase alleviates positive
supercoiling.
Origin
Single-strand
binding protein
• Single-strand binding proteins keep
the parental strands apart.
DNA helicase
3′
5′
DNA polymerase III
Topoisomerase II
• Primase synthesizes an RNA
primer.
• DNA polymerase III synthesizes a
daughter strand of DNA.
• DNA polymerase I excises the
RNA primers and fills in with
DNA (not shown).
Leading
strand
RNA primer
RNA
primer
DNA polymerase III
Replication fork
Okazaki fragment
Primase
5′
3′
Lagging strand
Parental DNA
• DNA ligase covalently links the
Okazaki fragments together.
DNA
ligase
3′
5′
Direction of fork movement
Brooker, fig 13.7
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Linked Okazaki
fragments
E. coli
chromosome
Origin of Replication
oriC
AT-rich region
5′ –GGA T CC T GGGT A T T AAAAAGAAGA T C T AT T T A T TT AGAGA T C T G T T C T AT
CC T AGGACCC AT A AT T T T T C T T C T AGAT AA AT AAAT CTCT AGAC AAGAT A
1
50
DnaA box
T G T GA T C T CT T A T T AGGAT CGC AC T GCCCT GT GGA T AACA AGGA T CGGCT
AC AC T AGAGA A T A ATCCT AGCGT GACGGGACACCT AT TGT T CC T AGCCGA
51
100
DnaA box
T T T A AGA T CA ACA ACCTGGA AAGGA T C AT T AACTG T GAAT GA T CGG T GAT
A A AT T C T AGT T GT T GGACC T T T CC T AGT AAT T GAC ACT T AC T AGCC AC T A
101
150
DnaA box
CC T GGACCGT A T A AGCTGGGA T C AGA A TGAGGGT T A TACA CAGC TC A A AA
GGACC T GGCA T AT T CGACCC T AGT C T T ACT CCCAAT ATGT GT CGAGT T T T
151
200
DnaA box
AC T GA AC AACGGT T GT TCT T TGGA T A ACTACCGGT T GA T CCA AGCT T CCT
T GAC T T GT T GCC A ACAAGA A ACCT AT T GAT GGCCA ACT AGGT T CGA AGGA
201
250
DnaA box
Brooker, fig 13.5
GAC AGAGT TA T CCA CAGTAGA T CGC –3′
CT GT C T C A AT AGGT GTCAT C T AGC G
251
275
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3′
5′
5′
3′
AT-rich region
DnaA boxes
DnaA proteins bind to DnaA boxes and to
each other. Additional proteins that cause
the DNA to bend also bind (not shown).
This causes the region to wrap around
the DnaA proteins and separates the
AT-rich region.
How the origin
sequence initiates
replication
DnaA protein
ATrich
region
5′
3′
3′
5′
DNA helicase (DnaB protein) binds to the
origin. DnaC protein (not shown) assists
this process.
DNA helicase
5′
3′
3′
5′
DNA helicase separates the DNA in both
directions, creating 2 replication forks.
3′
5′
3′
Brooker fig 13.6
Fork
Fork
5′
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Replication initiation cont.
Travels along the DNA
in the 5’ to 3’ direction
DNA helicase
5′
3′
3′
5′
DNA helicase separates the DNA in both
directions, creating 2 replication forks.
3′
5′
3′
Fork
Fork
5′
Bidirectional
replication
Brooker fig 13.6
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Schematic side view of DNA polymerase III
(bacterial)
DNA polymerase
catalytic site
Thumb
3′ exonuclease
site
3′
5′
Fingers
3′
Palm
5′
Template
strand
Incoming
DNA nucleotides (triphosphates)
(dNTPs)
Brooker, fig 13.8
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Model for how the leading strand and lagging strand
coordinate at the replication fork
Replisome
Leading strand
3′
Replication
DNA helicase
fork
Topoisomerase
3′
5′
Single-strand
binding proteins
5′
DNA
polymerase III
Primosome
Region
where
next Okazaki
fragment
will be made
Primase
RNA primer
5′
5′
5′
3′
New Okazaki
fragment
Older Okazaki
fragment
Brooker Fig 13.12
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Orientation of lagging strand in the replication bubble
Eukaryotes
have hundreds
of origins of
replication on
their (linear)
chromosome
Chromosome
Sister chromatids
Origin
Origin
Origin
Centromere
(DNA under the
kinetochore)
Origin
Origin
Before S phase
Brooker, ch 13
During S phase
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End of S phase
Replicating DNA of Eukaryotic Chromosomes (Drosophila
melanogaster)
Fig from iGenetics, Russell
Bacteria only have one origin on their
(circular) chromosome
Origin of
replication
Site where
replication
ends
(a) Bacterial chromosome replication
Replication
fork
Replication
fork
Brooker,
fig 13.4a
From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963).
Copyright holder is Cold Spring Habour Laboratory Press.
0.25 μm
(b) Autoradiograph of an E. coli chromosome in the act of replication
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Replication
forks
Replication rate
• Eukaryotic DNA replication
–
–
–
–
–
Typical human chromosome length: 100 million bp
Time to replicate a chromosome: minutes to hours
Hundreds of origins per chromosome
Replicon = ~20,000 to 300,000 bp long
500-5000 bp / minute at each replication fork
(slower than bacterial replication; that much harder
to “unwind” the DNA for replication).
• Bacterial (prokaryotic) replication:
– Single circular chromosome (~4.6 million base pairs
[bp])
– Single origin of replication  single replicon
(“Replication Bubble”)
Requirements of DNA Replication in a complex
organism
• Very low error rate:
– One human cell: 6 billion bp of DNA. A
copying error rate of 1 error/million nt
6000 errors with every cell division
• Very fast copy rate
– E. coli –1000 nt per minute  3 days to
replicate (real life: 20 minutes per cell
cycle; 1000 nt per second)
Linear chromosomes (eukaryotic) cannot easily replicate
the ends of chromosomes
No place for
a primer
3′
5′
Brooker, fig 13.21
DNA polymerase cannot link
these two nucleotides together
without a primer.
Linear chromosomes (eukaryotic) must fill in
gap left by RNA primer
Chromosome gets
shorter at the
telomeres with each
replication if
overhang is left.
Telomeric repeat sequences
3′
T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG
A A TCCCA A TCCCAA TCCCA A TCCCAA TCCCAA TCCCA A TCCCAA T
5′
In humans and most complex organisms, telomerase is only used in
continuously dividing stem cells (e.g. spermatogonia stem cells) 
most cells get shorter telomeres over time (age). What happened to
Dolly, the cloned sheep? (she was generated from a skin cell with
shorter telomeres, and she aged early)
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Overhang
Brooker, fig 13.20
How telomerase
“finishes” the
replication of linear
chromosomes
Telomere
5′
3′
3′
5′
Eukaryotic
chromosome
Repeat unit
The bindingpolymerizationtranslocation cycle
occurs many times
T T A G G GT T A GG G T T A G GG T TA G G G 3
CA AU C
A A T C C CAA T
Step 1 Binding
RNA
3′
5′
Telomerase synthesizes
a 6-nucleotide repeat.
Telomerase
G
G
T T A G G GT T A GG G T T A G GG T T A G G GT T A G
Step 2 Polymerization
CAAU C
A A T C C CAA T
This greatly
lengthens one of
the strands
Telomerase moves 6
nucleotides to the right and
begins to make another repeat.
T
T
Step 3 Translocation
T T A G G GT T A GG G T T A G GG T T A G G GT T A G G G
CA A U C
A A T C C CAA T
The complementary
strand is made by primase,
DNA polymerase, and ligase.
T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G 3′
A A T C C CAA T C CC A A T C C C A AT C C C A AU C CC A A U
Brooker, figure 13.22
RNA primer
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The end is now
lengthened
Go over lecture outline at end of
lecture
Concept Checks
• In the Meselson and Stahl experiment,
how was switching the bacterial media
from N15 to N14 important for supporting
the Semi-conservative model?
Concept check
• What are the functions of the A-T rich
region and DNA boxes in the Origin of
Replication?
Concept Check
• Why is primase needed for DNA
replication?
• Is the template strand read in the 5’ to 3’
direction or the 3’ to 5’ direction?
Concept Check
• Describe the differences between Dna
synthesis in the leading strand vs. the
lagging strand.
Which component functions immediately
after ligase?
a. Helicase
b. DNA Polymerase 1
c. DNA Polymerase 3
d. primase
e. none of the above
Which component functions immediately
after ligase?
a. Helicase
b. DNA Polymerase 1
c. DNA Polymerase 3
d. primase
e. none of the above