Week 3: 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, JD and Crick, FHC Nature 171 737-738 (1953)

 Proposed models of DNA replication

• In the late 1950s, three different mechanisms were

proposed for the replication of DNA

– Conservative model
• Both parental strands stay together after DNA replication

– Semiconservative model
• The double-stranded DNA contains one parental and one daughter
strand following replication

– Dispersive model
• Parental and daughter DNA are interspersed in both strands following
replication
 Proposed models of DNA replication

Original
double
helix

First round of
replication

Second round
of replication

(a) Conservative (b) Semiconservative (c) Dispersive

model model model

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• Matthew Meselson and Franklin Stahl devised an
experiment to distinguish between these models

– An ultracentrifuge can separate heavy and light molecules

by CsCl density-gradient centrifugation.
– Make all the DNA in E. coli heavy by growing them in 15N
medium for many generations – all nitrogen in DNA is 15N
rather than the normal 14N.
– Switch the E. coli to 14N medium
– Analyze DNA after each of several generations– “old”
DNA will have 15N and “new” DNA will contain 14N
• 14
N DNA-light
• 15
N/14N DNA-half heavy
• 15
N DNA-heavy
 Matthew Meselson and Franklin Stahl devised an
experiment to distinguish between these models

Result:

* Generation 1, 100%
half-heavy
 Semi-conservative
or dispersive
#
# Generation 2, 50% *
half-heavy, 50% light
 Semi-conservative

Wikipedia commons
 Properties of DNA replication:

1. DNA strands are antiparallel and complementary,

DNA replication must produce strands that are also
antiparallel and complementary.
2. The process must be extremely accurate. (Why?)
3. In addition to template DNA, E. coli DNA replication
requires:
a. Enzymes: helicase, primase, DNA polymerase III, DNA
polymerase I, DNA ligase, and topoisomerase
b. Additional proteins such as single strand binding proteins
(SSBs)
c. Deoxynucleotide triphosphates (dNTPs) and nucleotide
triphosphates (NTPs)
We can reconstitute a replication system in vitro
• Add template DNA – must contain a replication origin
• Proteins (enzymes) – from previous slide (see textbook)
• dNTPs (DNA building blocks and energy)
• NTPs (RNA building blocks to prime the system)

Some lessons learned

• Small genomes have a single replication origin
• Mammalian genomes have 50-100,000 origins
• Common replication mechanisms are used across all
organisms
• The rate of synthesis in eukaryotes is ~ 50 bp/sec
 DNA Polymerase
activity
NEW
STRAND

Notes:
• New synthesis retains anti-parallel strands.
• Synthesis is ALWAYS 5’ new base to 3’ of
existing base. ALWAYS...really, ALWAYS.
• dNTP is a building block AND the energy source
for the enzyme (polymerase) catalyzed reaction.
• The same thing is happening on the other template strand
Figure 5-3 Molecular Biology of the Cell (© Garland Science 2008)
 Direction of DNA Synthesis

As the replication fork moves, both strands are templates but synthesis
proceeds as a LEADING STRAND and a LAGGING STRAND

Figure 5-7 Molecular Biology of the Cell (© Garland Science 2008)

Other proteins required for DNA replication

So, let’s discuss the individual

proteins involved in initiating,
elongating, proofreading, and
terminating replication
When copying DNA, there is a
machinery that does this very
accurately
(although it is not perfect)
DNA Primase – DNA replication starts with RNA!

DNA Primase is an RNA polymerase. It initiates DNA replication by laying

down an RNA primer.
Helicase unwinds the parental DNA

ATP ADP + Pi
Topoisomerase

ATP ADP + Pi

This enzyme takes the supercoils out of the parental DNA molecule
The two DNA strands cannot be replicated in the same way

DNA polymerase can only synthesize DNA in the 5’

to 3’ direction:

For the ‘leading strand’, synthesis goes in the same

direction as the replication fork is moving: no
problem here.

For the ‘lagging strand’, synthesis proceeds in

the opposite direction from fork movement. This
strand must be made as a series of fragments called
Okazaki fragments (1000-2000 bp in prokaryotes,
100-200 in eukaryotes.)
Leman and Noguchi Genes 2013, 4(1), 1-32
 Lagging strand replication

Sealing the fragments with

DNA ligase

Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)

Proof-reading activities of DNA Polymerases

E. coli DNA Polymerase III

This is the main DNA synthesis enzyme. It makes the leading strand, and the
Okazaki fragments.
It has high processivity, and a low error rate (1 in 105 bases)
It has 3’ to 5’ exonuclease activity, allowing it to correct 99% of those errors, for
an overall error rate of 1 in 107 bases.
It does NOT have 5’ to 3’ exonuclease activity, so it cannot remove the RNA
primers.

E. coli DNA Polymerase I

This the DNA repair polymerase.
It has 5’ to 3’ exonuclease activity, used to remove the RNA primers
It has 3’ to 5’ exonuclease activity
DNA Ligase - 1
DNA polymerase I leaves a gap in the sugar-phosphate backbone

*
 DNA Ligase - 2
A different enzyme, DNA ligase, is required to seal this gap.

Enz + ATP/NAD > E-AMP >

DNA Ligase - 3
 A prokaryotic replication fork

Note: DNA polymerase binds as a dimer

to ensure simultaneous rate of
replication at the fork

For a good animation, see this: https://www.youtube.com/watch?v=4jtmOZaIvS0

Obtained from http://www.nature.com/scitable/content/proteins-at-the-y-shaped-dna-replication-14266059

Original from © 2002 From Molecular Biology of the Cell, 4th Edition by Alberts et al. Reproduced with permission of
Garland Science/Taylor & Francis LLC.
 Origins of DNA Replication

Each strand must be copied exactly once.

The timing and location of replication initiation are carefully

controlled.

Replication begins at a special sequence called an origin:

• prokaryotic chromosomes have a single origin
• eukaryotic chromosomes have multiple origins

From the origin, replication proceeds bidirectionally

• this yields two replication forks where DNA is synthesized.
 A replication origin generates two replication forks

https://quizlet.com/75944826/biology-test-2-flash-cards/
Bacterial chromosomes have a single replication origin

Origin of
replication

Replication
forks

Site where
replication
ends

(a) Bacterial chromosome replication

Replication
fork

Replication
fork

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

From SLideShare.net Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Linear chromosomes of eukaryotes are replicated from
multiple origins
E. coli
Special DNA sequences are found at the
chromosome origin of replication (A-T rich)
oriC

AT-rich region
5′ –GGA T CC TGGG T A T T AAAAAGAAGA T C T A T T TA T T T AGAGA T C TG T T C T A T
CC T AGGACCC A T A A T T T T T C T T C T AGA T AA AT AAA T CTCT AGAC AAGA T A
1 DnaA box 50
TG TGA TC T CT T A T T AGGA T CGC A C TGCCCTG T GGA T AACA AGGA T CGGCT
AC AC T AGAGA A T A A TCCT AGCGT GACGGGACACCT A T TGT T CC T AGCCGA
51 DnaA box 100
T T T A AGA TCA A CA ACCTGGA AAGGA T C AT T AA CTG TGAA TGA T CGG TGA T
A A A T T C T AGT T GT T GGACC T T T CC T AGT AA T T GAC ACT T AC T AGCC AC T A
101 DnaA box 150
CC TGGA CCGT A T A AGCTGGGA T C AGA A TGAGGGT T A TACA CAGC TC A A AA
GGACC T GGCA T A T T CGACCC T AGT C T T ACT CCCAA T ATGT GT CGAGT T T T
151 DnaA box 200
A C TGA A C AACGG T TGT TCT T TGGA T A ACTACCGGT TGA T CCA AGCT T CCT
T GAC T T GT TGCC A ACAAGA A ACCT A T T GAT GGCCA ACT AGGT T CGA AGGA
201 DnaA box 250
GA C AGAG T TA T CCA CAGTAGA TCGC –3′
CT GT C T C A AT AGGT GTCAT C T AGC G
251 275
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Initiation of DNA replication by DnaA and
helicase 5′
3′
3′
5′

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.

AT- DnaA protein

rich
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′ Fork Fork
3′ 5′
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required for reproduction or display.
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

DNA helicase
Travels along the
DNA in the 5’ to 3’
direction

5′ 3′
3′
5′

DNA helicase separates the DNA in both

directions, creating 2 replication forks.

3′
Fork Fork
5′
3′ 5′

Bidirectional
replication
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 DNA Polymerases of eukaryotes

• Mammalian cells contain well over a dozen different

DNA polymerases (although many share protein
subunits)

• Four: alpha (α), delta (), epsilon () and gamma ()
have the primary function of replicating DNA
• α,  and   Nuclear DNA
•   Mitochondrial DNA
 Polymerase switching in eukaryotes

• DNA pol α is the only polymerase to associate with primase

– The DNA pol α/primase complex synthesizes a short RNA-
DNA hybrid

– This is used by DNA pol  or  for the processive elongation

of the leading and lagging strands

• The exchange of DNA pol α for  or  is called a polymerase

switch
– It occurs only after the RNA-DNA hybrid is made
A special mechanism is needed to replicate the ends of
chromosomes: telomerase

DNA polymerase cannot link

these two nucleotides together
without a primer.
3′

No place
for a
primer

5′

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 The DNA at telomeres is composed of a simple
repeated sequence.

Telomeric repeat sequences

3′

T T T T T T T T T T T T T T T T
AG GG A GG G A GG G A G GG A GG G A GGG A G GG A G GG
A A A A A A A A A A A A
T C C C T C C C T C C C T C C C T C C C T
Overhang
5′

A special DNA polymerase called telomerase carries an internal

RNA template that directs the synthesis of these repeats.
 Telomere

Telomerase 3′
5′
5′
3′

activity Eukaryotic
chromosome

Repeat unit

T T AGGG T T A GGG T T A GGG T TAGGG 3

CC CA AU CCC
Step 1: Binding
A A TC CCAA T
RNA
3′ 5′
Telomerase synthesizes
a 6-nucleotide repeat.
Telomerase
G
G
T T AGGG T T A GGG T T A GGG T T A GGG T T A G
CC C A A U CCC
Step 2: Polymerization
A A TC CCAA T

The binding- Telomerase moves 6

polymerization- nucleotides to the right and
begins to make another repeat.

translocation cycle T
T

can occur many

T T AGGG T T A GGG T T A GGG T T A GGG T TA G GG
C C C A A U CCC
Step 3: Translocation
A A TC CCAA T
times
The complementary
strand is made by primase,
DNA polymerase, and ligase.
A primer can now
This greatly
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′ bind for synthesis of
lengthens the
A A T C C CAA T C C C A A T C C C A A T C C C A AU C CC A A U more telomeric
telomere RNA primer sequence
 Telomerase activity: Just the nucleic acids

1. The guide RNA anneals to the end of the last DNA repeat
5’TTAGGG
3’CCCAAUCCC
2. The DNA polymerase activity of telomerase synthesizes another DNA repeat

5’TTAGGGTTAGGG
3’CCCAAUCCC
3. The guide RNA (and the telomerase enzyme) moves over by one repeat

5’TTAGGGTTAGGG
3’CCCAAUCCC
Steps 2 and 3 repeat

This is an example of NON-TEMPLATE DRIVEN BASE ADDITIONS

 Telomerase activity is important in cancer

Normal cells grown in culture stop growing after finite number of cell
divisions (25-50 divisions) and die soon thereafter

Telomere shortening occurs at each doubling

Cancer cells grow indefinitely because

Telomerase is functional in these cells
Essential components of a eukaryotic chromosome
The DNA in eukaryotic chromosomes is
organized by wrapping around histones
 Genetic variation in the population

DNA sequence Biological trait (Genotype)

(Phenotype)
Environment

ATTCGCATGGACC (part of 3 billion bp)

SNP 1 C
SNP 2 A

SNPs Single Nucleotide Polymorphisms Some affect phenotype

CNVs Copy Number Variations Some are useful markers
 What have we learned from the human genome?

1. The entire 6 x 109 bp sequence codes for ~17,000

genes.
2. ~ 2% codes for proteins
3. Lots of repetitive DNA
4. You and the person next to you share about 99.9% sequence identity.
(= million differences)
5. In a population variation occurs about 1/300-1,000 bp (SNPs)
6. Genotype determines phenotype (trait) = individually unique!
All this means the variation in the genome will likely have an
impact on disease risk, progression, response.
Good reason to think about personalized medicine!