BIO101 CAPE BIOLOGY

UNIT 1
DNA Replication
Syllabus Objectives:

• Explain the importance of hydrogen bonds and base pairing in

DNA replication;
• Describe in words and diagrams how DNA replicates
• Recognize that the process of DNA replication is facilitated by
enzymes
• Recognize the significance of 5’ to 3’ directionality
• State advantages of Semiconservative replication
• Explain how DNA replication facilitates life
Lecture Outline:

• Review structure of nucleic acids & Review principle

of complimentary base pairing

• Process of DNA replication

• Semiconservative replication
• Uncoiling, Complimentary Base pairing, Polymerization,
Recoiling
REVIEW
Double helix
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5´
Phosphate group
• 2 strands are polymers of
P
5
O
nucleotides
1

• Phosphodiester backbone
4 Phosphodiester bond
3 2

P 5
– repeating sugar and
4
O
1 phosphate units joined by
3 2
phosphodiester bonds
• Wrap around 1 axis
P 5
O
4 1
5-carbon sugar
3 2
Nitrogenous base
• Antiparallel
P 5
O
1
4
3
2
OH

3
6
Antiparallel Nature of DNA

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2nm
5′ 3′

T A

G 3.4nm
C

Minor T
groove

A T
0.34nm

G C

Major
groove
G

A T

G C

Major
groove

Minor
groove

9
5′
3′
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Hydrogen
bond H
H N O H N H
• Complementarity of bases
N
• A forms 2 hydrogen bonds
G N H N C H
Sugar N N
with T N H
Sugar

• G forms 3 hydrogen bonds

H

Hydrogen
with C H bond

H O CH3
• Gives consistent diameter
H N N

N A N H N T H
Sugar N N
H
Sugar

10
DNA REPLICATION
 DNA Replication in Eukaryotic Cells

• All cells carry a full complement of hereditary material

(except mature erythrocytes; but the immature reticulocytes do).

• As organisms grow and new cells are formed, exact

copies of the DNA must be generated for each
cell.

• DNA is replicated in interphase before cell division

(mitosis or meiosis) so daughter cells can obtain genetic
material of the organism.
1
2
DNA Replication

• During replication, the sequence of nitrogenous bases and their

pairing scheme (A-T, C-G) on the DNA must be kept exact and
specific so the correct information will be transmitted from one
cell to the next.

• Type of replication: Semiconservative

✓ Each of the new DNA molecules is made of one OLD strand

and one NEW strand.
1
3
DNA Replication
DNA Replication

• DNA replication is semi-conservative:

• Each strand of the original DNA double helix has been
used as a template for construction of a new strand
identical to its former partner.
• The result is 2 identical double helices, each with same information as the
original DNA but formed by one original strand and one newly synthesized
strand.

15
 The Basic Principle: Base Pairing to a
Template Strand

• Since the two strands of DNA are complementary,

each strand acts as a template for building a new
strand in replication
• In DNA replication, the parent molecule unwinds,
and two new daughter strands are built based on
base-pairing rules

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• Watson and Crick’s semiconservative model of
replication predicts that when a double helix replicates,
each daughter molecule will have one old strand (derived
or “conserved” from the parent molecule) and one newly
made strand
• Competing models were the conservative model (the two
parent strands rejoin) and the dispersive model (each
strand is a mix of old and new)
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 First Second
Parent cell replication replication

Figure 13.12 (a) Conservative model

(b) Semiconservative model

(c) Dispersive model

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Meselson and Stahl – 1958

• Experiments by Matthew Meselson and

Franklin Stahl supported the semiconservative
model

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Meselson and Stahl – 1958

• Bacterial cells were grown in a heavy isotope of

nitrogen, 15N
• All the DNA incorporated 15N
• Cells were switched to media containing lighter 14N
• DNA was extracted from the cells at various time
intervals

20
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DNA
E. coli
E. coli cells grown
15N medium in 15N medium

Cells shifted to
14N medium 14N medium and

allowed to grow

Samples taken at
three time points
and suspended in
cesium chloride
solution
0 min 20 min 40 min
0 rounds 1 round 2 rounds

Samples are centrifuged

0 rounds 1 round 2 rounds

Top Bottom Rounds of 21

replication
From M. Meselson and F.W. Stahl/PNAS 44(1958):671
 Experiment
Bacteria Bacteria
cultured in transferred
medium to medium
with15N with14N
(heavy (lighter
Figure 13.13
isotope) isotope)

Results DNA sample DNA sample Less

centrifuged centrifuged dense
after first after second More
replication replication dense

Conclusion
Predictions: First replication Second replication

Conservative
model

Semiconservative
model

Dispersive
model
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Meselson and Stahl’s Results

• Conservative model = rejected

• 2 densities were not observed after round 1
• Semiconservative model = supported
• Consistent with all observations
• 1 band after round 1
• 2 bands after round 2
• Dispersive model = rejected
• 1st round results consistent
• 2nd round – did not observe 1 band
23
 Experiment
Figure 13.13-1
Bacteria Bacteria
cultured in transferred
medium to medium
with15N with14N
(heavy (lighter
isotope) isotope)

Results DNA sample DNA sample Less

centrifuged centrifuged dense
after first after second
More
replication replication
dense

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 Conclusion

Predictions: First replication Second replication

Figure 13.13-2

Conservative
model

Semiconservative
model

Dispersive
model

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DNA Replication

• Requires 3 things
• Something to copy
• Parental DNA molecule
• Something to do the copying
• Enzymes
• Building blocks to make copy
• Nucleotide triphosphates

26
DNA Replication

•DNA replication includes

• Initiation – replication begins
• Elongation – new strands of DNA are
synthesized by DNA polymerase
• Termination – replication is terminated

27
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Template Strand New Strand Template Strand New Strand

HO 3′ 5′ HO 3′ 5′

C P C P
G G
O O
O O
Sugar–
phosphate P P
backbone
T P T P
A O A O
O O
P P

P P
A DNA polymerase III A
T O T O
O O
P P

P P
C C
G O G O
O O
P P
3′ P
OH P P
A A O
T Pyrophosphate
O O
T P P P
P P
O
3′ OH
A A
O O
OH
P P
5′ 5′

28
DNA Replication
 DNA Replication: A Closer Look

• The copying of DNA is remarkable in its speed and

accuracy
• More than a dozen enzymes and other proteins participate
in DNA replication
• Much more is known about how this “replication
machine” works in bacteria than in eukaryotes
• Most of the process is similar between prokaryotes and
eukaryotes

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DNA Replication
• Main steps.
1. Uncoiling of the double helix
2. Complementary base pairing on both strands
3. Polymerization and coiling
 DNA Replication
• Process involves enzymes

9/24/2015 BIOL00112015-16 5
33
DNA Replication Summary

1. Uncoiling
• Double helix uncoil.
• Two strands of
polymerised nucleotides
separate into individual Replication fork
DNA strands by
successively breaking
hydrogen bonds between
the base pairs.

34
 Getting Started

• Replication begins at sites called origins of replication,

where the two DNA strands are separated, opening up a
replication “bubble”
• At each end of a bubble is a replication fork, a
Y-shaped region where the parental strands of DNA are
being unwound

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 • A eukaryotic chromosome
• May have hundreds or even thousands of replication
origins
Origin of replication Parental (template) strand
0.25 µm
Daughter (new) strand

1 Replication begins at specific sites

where the two parental strands
separate and form replication
bubbles. Bubble Replication fork

2 The bubbles expand laterally, as

DNA replication proceeds in both
directions.

3 Eventually, the replication

bubbles fuse, and synthesis of
the daughter strands is
complete. Two daughter DNA molecules

(a) In eukaryotes, DNA replication begins at many sites along the giant (b) In this micrograph, three replication
DNA molecule of each chromosome. bubbles are visible along the DNA of
Figure 16.12 a, b a cultured Chinese hamster cell (TEM).
Figure 13.14

Primase

Topoisomerase
3
RNA
5
3 primer
5
Replication
3 fork

5
Helicase
Single-strand binding
proteins

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Proteins and Enzymes That Assist in DNA
Replication

• Helicases are enzymes that untwist the double

helix at the replication forks
• Single-strand binding proteins bind to and
stabilize single-stranded DNA
• Topoisomerase relieves the strain caused by tight
twisting ahead of the replication fork by breaking,
swiveling, and rejoining DNA strands
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DNA Replication

2. Complimentary Base pairing

• As a segment of unwound DNA is
exposed, free nucleotides from the
surroundings encounter it and align with
their complementary bases
• They bind with H bonds, A=T and CG.

39
DNA Replication

1. Unwinding

2. Complimentary
base pairing

40
DNA Replication

• New strands are both synthesised from 5’ to 3’ ends.

• So the two new strands do not run in the same

direction.

• In one strand, synthesis is continuous (leading strand).

• In the other strand, synthesis is done in sections

(lagging strand).
Elongating a New DNA Strand
• Elongation of new DNA at a replication fork
• Is catalyzed by enzymes called DNA polymerases, which add nucleotides
to the 3 end of a growing strand
New strand Template strand
5 end 3 end 5 end 3 end

Sugar A T A T
Phosphate Base

C G C G

G C G C

A T A
P P OH
C Pyrophosphate 3 end C
OH
Nucleoside 2 P
triphosphate 5 end 5 end
Figure 16.13
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5 5 3

3 5 3 5

RNA polymerase makes primer DNA polymerase extends primer

• DNA polymerase
• Matches existing DNA bases with complementary
nucleotides and links them
• All have several common features
• Add new bases to 3′ end of existing strands
• Synthesize in 5′-to-3′ direction
• Requires a primer of RNA
43
Antiparallel Elongation

• How does the antiparallel structure of the double

helix affect replication?
 Antiparallel Elongation

• Newly replicated DNA strands must be formed

antiparallel to the template strand
• DNA polymerases add nucleotides only to the free
3 end of a growing strand; therefore, a new DNA
strand can elongate only in the 5 to 3 direction

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• Each nucleotide that is added to a growing DNA consists of a
sugar attached to a base and to three phosphate groups
• dATP is used to make DNA and is similar to the ATP of
energy metabolism
• The difference is in the sugars: dATP has deoxyribose, while
ATP has ribose
• As each monomer nucleotide joins the DNA strand, it loses
two phosphate groups as a molecule of pyrophosphate
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Figure 13.16
New strand Template strand
5 3 5 3

Sugar A T A T
Phosphate Base

C G C G

G C DNA G C
poly-
merase
3 A T A

P Pi
C 3 C
Pyro-
phosphate
Nucleotide
5 5
2 Pi
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• DNA polymerases add nucleotides
• Only to the free 3 end of a growing strand
• Along one template strand of DNA, the leading strand
• DNA polymerase III can synthesize a complementary strand continuously, moving
toward the replication fork

• To elongate the other new strand of DNA, the lagging strand

• DNA polymerase III must work in the direction away from the replication fork
• The lagging strand
• Is synthesized as a series of segments called Okazaki fragments, which are then
joined together by DNA ligase
• Synthesis of leading and lagging strands
during DNA replication
1 DNA pol Ill elongates
DNA strands only in the
5 3 direction. 3 2 One new strand, the leading strand,
Parental DNA 5 can elongate continuously 5 3
as the replication fork progresses.
5
3 Okazaki
3 The other new strand, the
fragments
lagging strand must grow in an overall
2 3 5 direction by addition of short
1 3
segments, Okazaki fragments, that grow
5 5 3 (numbered here in the order
DNA pol III they were made).

Template
strand

4 DNA ligase joins Okazaki

fragments by forming a bond between
Leading strand their free ends. This results in a
Lagging strand
3 continuous strand.
2 1

Template
strand DNA ligase
Figure 16.14 Overall direction of replication
Priming DNA Synthesis

• DNA polymerases cannot initiate the synthesis of a

polynucleotide
• They can only add nucleotides to the 3 end
• The initial nucleotide strand
• Is an RNA or DNA primer

• Only one primer is needed for synthesis of the leading strand

• But for synthesis of the lagging strand, each Okazaki fragment must
be primed separately
• The enzyme, primase (a type of RNA
Polymerase), starts an RNA chain with a single
RNA nucleotide and adds RNA nucleotides one at a
time using the parental DNA as a template
• The primer is short (5–10 nucleotides long)
• The new DNA strand will start from the 3 end of
the RNA primer
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•Along one template strand of DNA, the
DNA polymerase synthesizes a leading
strand continuously, moving toward the
replication fork

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 Leading Overview
Origin of replication Lagging
strand strand
Figure 13.17

Primer

Leading
Lagging strand Overall strand
directions
of replication

Origin of replication
3
5
RNA primer
5 3 Sliding clamp
3
DNA pol III
5
Parental DNA
3
5

5 Continuous elongation
3 3 in the 5 to 3 direction

5 Inc.
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 Origin of replication
Figure 13.17-2
3
5
RNA primer
5 3 Sliding clamp
3
DNA pol III
5
Parental DNA
3
5

5 Continuous elongation
3 3 in the 5 to 3 direction

5
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• To elongate the other new strand, the lagging
strand, DNA polymerase must work in the
direction away from the replication fork
• The lagging strand is synthesized as a series of
segments called Okazaki fragments

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• After formation of Okazaki fragments, DNA
polymerase I removes the RNA primers and
replaces the nucleotides with DNA
• The remaining gaps are joined together by
DNA ligase

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1 Primase joins RNA nucleotides
into a primer. 3
5

5 3

Template
2 DNA pol III adds DNA nucleotides to the
strand primer, forming an Okazaki fragment.
3 RNA primer 3 5
1
5

3 After reaching the next

RNA primer (not shown),
DNA pol III falls off.
Okazaki 3
3 fragment
5
1
5
4 After the second fragment is
primed. DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off. 5
3
3 2 5
1

5 DNA pol 1 replaces the

RNA with DNA, adding to
the 3 end of fragment 2.
5 3
3
2 5
1

6 DNA ligase forms a bond 7 The lagging strand

between the newest DNA in this region is now
and the adjacent DNA of complete.
fragment 1. 5 3
3
2 5
1

Figure 16.15 Overall direction of replication

DNA Replication
 DNA Replication

3. Polymerization.
• Individual nucleotides are then joined to each other by
phosphodiester bonds between a phosphate on C5 (5’
end) of one nucleotide and the 3' OH group (C 3) of the
next nucleotide.

• 5’ to 3’ direction so nucleotide added to 3’end of

polymer

59
 DNA Replication
3. Polymerization

deoxyadenosine
monophosphate

deoxythymidine
monophosphate
60
DNA Replication

61
 Overview
Lagging Origin of replication
Figure 13.18 strand
Leading Lagging
strand strand

Leading RNA primer

Overall directions strand for fragment 2
of replication 5 Okazaki
3 DNA pol III
3 Primase makes fragment 2
Primer for makes Okazaki
RNA primer. Origin of leading fragment 2.
replication strand
5 3 3
Template 5 3 5
strand 5
5
DNA pol III 3 DNA pol I
3 RNA primer makes Okazaki replaces RNA
for fragment 1 fragment 1. with DNA.
3
5 3 5 3 5
5 DNA ligase forms
bonds between
DNA pol III 5
DNA fragments.
detaches. 3
3
Okazaki
fragment 1 3
5 5
3
5
© 2016 Pearson Education, Inc. Overall direction of replication
Figure 13.18-2-s1 3 Primase makes Primer for
RNA primer. Origin of leading
replication strand
5 3
Template 5 3
strand 5

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Figure 13.18-2-s2 3 Primase makes Primer for
RNA primer. Origin of leading
replication strand
5 3
Template 5 3
strand 5

DNA pol III

3 RNA primer makes Okazaki
for fragment 1 fragment 1.

5 3 5 3
5

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 Primase makes Primer for
Figure 13.18-2-s3 3
RNA primer. Origin of leading
replication strand
5 3
Template 5 3
strand 5

DNA pol III

3 RNA primer makes Okazaki
for fragment 1 fragment 1.

5 3 5 3
5

DNA pol III

detaches.
3
Okazaki
fragment 1
5
3
5
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 RNA primer for fragment 2

Figure 13.18-3-s1 5 Okazaki

DNA pol III
3 fragment 2
makes Okazaki
fragment 2.

3
5

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 RNA primer for fragment 2
Figure 13.18-3-s2 5 Okazaki
DNA pol III
3 fragment 2
makes Okazaki
fragment 2.

3
5
5
3 DNA pol I
replaces RNA
with DNA.

3
5

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 RNA primer for fragment 2

Figure 13.18-3-s3 5 Okazaki

DNA pol III
3 fragment 2
makes Okazaki
fragment 2.

3
5
5
3 DNA pol I
replaces RNA
with DNA.

3
5
DNA ligase forms
bonds between
5 DNA fragments.
3

3
5
Overall direction of replication
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DNA Replication
DNA Replication

• During cell division each of these DNA copies will

become part of one of the daughter cells.
• Each step in this process is assisted and
controlled by enzymes
• There is also proofreading so that mismatched
base pairs (eg. A-G pair) are excised and repaired.

71
Other Proteins That Assist DNA Replication

• Helicase, topoisomerase, single-strand binding protein

• Are all proteins that assist DNA replication

Table 16.1
 • A summary of DNA replication

Overall direction of replication Lagging

Leading
1 Helicase unwinds the strand Origin of replication strand
parental double helix.
2 Molecules of single- 3 The leading strand is
strand binding protein synthesized continuously in the
stabilize the unwound 5→ 3 direction by DNA pol III. Lagging Leading
template strands.
strand OVERVIEW strand
DNA pol III

Leading
strand

5 Replication fork
DNA pol I DNA ligase
3
Primase 2
Parental DNA DNA pol III Lagging 1
Primer strand 3
4 Primase begins synthesis
3 5
of RNA primer for fifth 4
Okazaki fragment.

5 DNA pol III is completing synthesis of 6 DNA pol I removes the primer from the 5 end 7 DNA ligase bonds
the fourth fragment, when it reaches the of the second fragment, replacing it with DNA the 3 end of the
RNA primer on the third fragment, it will nucleotides that it adds one by one to the 3 end second fragment to
dissociate, move to the replication fork, of the third fragment. The replacement of the the 5 end of the first
and add DNA nucleotides to the 3 end last RNA nucleotide with DNA leaves the sugar- fragment.
of the fifth fragment primer. phosphate backbone with a free 3 end.
Figure 16.16
74
DNA Replication

www.cstephenmurray.com

75
 Proofreading and Repairing DNA

• DNA polymerases proofread newly made DNA, replacing any

incorrect nucleotides
• In mismatch repair of DNA, other enzymes correct errors in
base pairing
• A hereditary defect in one such enzyme is associated with a form
of colon cancer
• This defect allows cancer-causing errors to accumulate in DNA
faster than normal

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• DNA can be damaged by exposure to harmful chemical or physical
agents such as cigarette smoke and X-rays
• It can also undergo spontaneous changes
• In nucleotide excision repair, a nuclease cuts out and
replaces damaged stretches of DNA

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 5 3

3 5

Nuclease
Figure 13.21-s1

5 3

3 5

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 5 3

3 5

Nuclease
Figure 13.21-s2

5 3

3 5

DNA
polymerase

5 3

3 5

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 5 3

3 5

Nuclease
Figure 13.21-s3

5 3

3 5

DNA
polymerase

5 3

3 5

DNA
ligase

5 3

3
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 Evolutionary Significance of Altered DNA
Nucleotides

• The error rate after proofreading repair is low but

not zero
• Sequence changes may become permanent and can
be passed on to the next generation
• These changes (mutations) are the source of the
genetic variation upon which natural selection
operates
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Protein Synthesis

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