CAMPBELL
BIOLOGY
DNA
TENTH
EDITION
Reece • Urry • Cain • Wasserman • Minorsky • Jackson
16
 Deoxyribonucleic acid – DNA
DNA: The Molecular Basis of Inheritance
 The blueprint to making proteins!!!
 Chromosomes located inside the nucleus
contains long coiled strands of DNA
Lecture Presentation by
Dr Burns
NVC Biol 120
© 2014 Pearson
Inc.
Copyright
© 2009Education,
Pearson Education,
Inc.
DNA’s Discovery
Watson and Crick
Rosalind Franklin →
The Players
 Crick: Ph.D. student at Cambridge in England
working on X-ray Crystallography of the protein
hemoglobin
 Watson: Young American scientist visiting the
lab to do some work on a protein
 Both were interested in unraveling the secret of
DNA’s structure – it was not what they were
supposed to be working on
 Wilkins: Working on DNA structure, crystallized
DNA fibers
 Franklin: Working at the same university as
Wilkins, just down the hall. Did the X-ray
Crystallography on Wilkins DNA fibers
 Linus Pauling: discovered the three dimensional
structure of proteins know as alpha helixes
 Chargaff: Discovered that A=T and G=C
Adenine levels always equal thymine levels,
Guanine levels always equal cytosine
1
 Watson and Crick put all the pieces of
information together.
 They built models to help them come up with the
structure.
 Franklin gave a talk describing her work with
the X-Ray Crystallography, Watson attended
but he was not the crystallographer and did
not see the implications of her work
 Watson and Crick met with Wilkins and he
shared Franklin’s work with both of them
(without her permission or knowledge)
 They knew it was a race so they published a one
page article in Nature with their ideas – they
performed no experiments but were able to see
the big picture
 Crick, Watson and Wilkins received the Nobel
Prize for their work. Rosalind received no credit
until much later. She died before the Nobel
Prize, the prize is not awarded after a person
has died
Figure 16.7
C
C
5′ end
G
Hydrogen bond
G
G
C
C
G
3′ end
A
T
3.4 nm
A
T
G
C
C
T
1 nm
A
G
C
G
A
G
A
T
T
3′ end
A
T
G
C
T
C
C
C
G
G
A
T
A
(a) Key features of
DNA structure
0.34 nm
5′ end
(b) Partial chemical structure
(c) Space-filling
model
Animation: Hershey-Chase Experiment
Right-click slide / select “Play”
© 2014 Pearson Education, Inc.
Fig. 14.4
Nucleotide Structure
© 2011 Pearson Education, Inc.
Figure 16.5
Sugar–phosphate
backbone
Nitrogenous bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Guanine (G)
DNA
nucleotide
Sugar
(deoxyribose)
3 end
Nitrogenous base
2
DNA Structure
Fig. 14.3-1
 Nucleotides that build DNA have:
 One phosphate (ATP has three)
 One sugar = deoxyribose
 One base.
 The nucleotides vary in the type of base – there
are four different bases in DNA: Adenine (A),
Thymine (T), Guanine (G), Cytosine (C)
 There is a 5’ end and a 3’ end
3
Animation: DNA and RNA Structure Rightclick slide / select “Play”
© 2011 Pearson Education, Inc.
Bonds
 The sugars and phosphates link together by
covalent bonds to form the rail on the outside
= phosphodiester linkage.
 The sugars are covalently bound to a base
 The complementary bases are attracted to
each other by hydrogen bonds
Double Helix
 Two strands bonded together by hydrogen
bonds between the bases = weak bonds
 Each strand has nucleotides bonded together
covalently by the phosphate and the sugar
 Base pairs are two nucleotides, one on each
complementary strand of a DNA molecule
Base Pairs
 The bases pair up in a specific manner:
 Adenine (A) pairs with Thymine (T)
 Guanine (G) pairs with Cytosine (C)
 Purines: Adenine and Guanine
 Pyrimidines: Thymine and Cytosine
4
Figure 16.8
 Remember that on one strand:
 The base is covalently bonded to the sugar,
which is covalently bonded to the phosphate
Sugar
Sugar
Adenine (A)
Thymine (T)
 Between the two strands the bases are
bonded together by hydrogen bond
 A–T
 C–G
Sugar
Sugar
Cytosine (C)
The bonds between the sugars and phosphates are
25%
Peptide
Phosphodiester
Hydrogen
Ionic
25%
e
Pe
id
pt
di
ho
25%
r
te
25%
n
ge
ro
es
ni
Io
d
Hy
p
os
Ph
25%
Peptide
Phosphodiester
Hydrogen
Ionic
c
e
Pe
id
pt
i
od
ph
os
Ph
25%
25%
r
te
25%
n
ge
ro
es
ni
Io
d
Hy
c
Guanine pairs with
33%
e
33%
Cy
to
sin
33%
e
1. Thymine
2. Adenine
3. Cytosine
nin
e
ne
an
i
Gu
Th
ym
in
33%
Cy
to
sin
33%
e
33%
Ad
e
Adenine pairs with
1. Thymine
2. Guanine
3. Cytosine
1.
2.
3.
4.
e
1.
2.
3.
4.
The bonds between the bases are
Th
ym
in
Guanine (G)
5
The bases are bound to
The bases are bound to the sugar by this kind of bond
1. Sugars
2. Phosphates
50%
50%
rs
ga
Su
1.
2.
3.
4.
Covalent
Phosphodiester
Hydrogen
Ionic
le
va
Co
s
te
ha
p
os
nt
i
od
ph
os
25%
25%
r
te
es
n
ge
ro
d
Hy
25%
ni
Io
c
Ph
Ph
DNA replication
The sugar in DNA is
1.
2.
3.
4.
25%
Ribose
Deoxyribose
Glucose
Cellulose
25%
25%
25%
25%
 The relationship between structure and function is
manifest in the double helix
 Watson and Crick noted that the specific base
pairing suggested a possible copying mechanism
for genetic material
e
os
b
Ri
e
os
ib
yr
ox
De
se
co
Glu
Ce
llu
se
lo
© 2011 Pearson Education, Inc.
DNA Replication
 When the structure of DNA was worked
out it became apparent how it happens
 It is semiconservative replication
 Each strand of DNA is the template for
building new complementary strands
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
© 2011 Pearson Education, Inc.
6
Animation: DNA Replication Overview
Right-click slide / select “Play”
© 2011 Pearson Education, Inc.
Figure 16.9-1
Figure 16.9-2
A
T
A
T
A
T
C
G
C
G
C
G
T
A
T
A
T
A
A
T
A
T
A
T
G
C
G
C
G
C
(a) Parent molecule
(a) Parent molecule
Figure 16.9-3
(b) Separation of
strands
Semiconservative model
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
 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)
© 2011 Pearson Education, Inc.
7
Parent
cell
Figure 16.10
First
replication
Second
replication
(a) Conservative
model
Fig. 14.13
(b) Semiconservative
model
(c) Dispersive model
DNA Replication: A Closer Look
BioFlix: DNA Replication
 The copying of DNA is remarkable in its speed
and accuracy
 More than a dozen enzymes and other proteins
participate in DNA replication
© 2011 Pearson Education, Inc.
Getting Started
 Replication begins at particular sites called origins
of replication, where the two DNA strands are
separated, opening up a replication “bubble”
 A eukaryotic chromosome may have hundreds or
even thousands of origins of replication
 Replication proceeds in both directions from each
origin, until the entire molecule is copied
 At the end of each replication bubble is a
replication fork, a Y-shaped region where new
DNA strands are elongating
Animation: Origins of Replication
Right-click slide / select “Play”
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.
8
Figure 16.12
(a) Origin of replication in an E. coli cell
Origin of
replication
Daughter (new)
strand
Doublestranded
DNA molecule
DNA
DNA
Replication
Replication
(b) Origins of replication in a eukaryotic cell
Double-stranded
DNA molecule
Origin of replication
Parental (template) strand
Replication
fork
Replication
bubble
Parental (template)
strand
1. An enzyme, helicase, unwinds the DNA
molecule and breaks the hydrogen bonds
between the base pairs
2. Single-strand binding proteins bind to each
strand and keep them from reforming the double
helix
3. Topoisomerases produce breaks in the DNA
molecule to relieve the stress of unwinding, then
they also repair these breaks.
Daughter (new)
strand
Replication fork
Bubble
Two daughter
DNA molecules
0.5 m
0.25 m
Two daughter DNA molecules
Figure 16.13
DNA Replication
Primase
3
Topoisomerase
3
5
5
RNA
primer
3
Helicase
5
Single-strand binding
proteins
 Now the complementary strand needs to be built:
4. Enzymes called DNA polymerases build the new
complementary strand by adding new nucleotides
to the 3’ end which pair with the old DNA.
5. But DNA polymerase can not start the process. A
primer of RNA bases is first built for the
complementary strand.
6. An enzyme called primase adds the RNA bases,
then DNA polymerase can take over and keep
building the complementary strand.
7. The primer is replaced by DNA bases
DNA
DNA
Replication
Replication
DNA
DNA
Replication
Replication
8. DNA polymerase builds the new
complementary strand from the 5’ end to the 3’,
by adding the nucleotides to the 3’ end =
leading strand
9. The other strand = lagging strand, is build in
short stretches going from 5’ to 3’
 But the other strand also needs to be replicated
but it can only build new strands by adding to
the 3’ end
10. The short strands being built are called
Okazaki fragments
11. DNA ligase join the Okazaki fragments
9
Energy to power building complementary strand
Synthesizing a New DNA Strand
 The incoming nucleotides have three
phosphates, only one is used to bond to the
sugar molecule
 Enzymes called DNA polymerases catalyze the
elongation of new DNA at a replication fork
 Most DNA polymerases require a primer and a DNA
template strand
 The rate of elongation is about 500 nucleotides per
second in bacteria and 50 per second in human
cells
 The energy needed to build the new DNA
strand comes from taking the other two
phosphates off.
© 2011 Pearson Education, Inc.
Figure 16.14
Antiparallel Elongation
Template strand
3
New strand
5
Sugar
Phosphate
5
3
A
Base
T
A
T
C
G
C
G
G
C
G
C
T
A
DNA
polymerase
OH
3
A
P
C
Nucleoside
triphosphate
OH
3
Pi
Pyrophosphate
 The antiparallel structure of the double helix
affects replication
 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
C
2Pi
5
5
© 2011 Pearson Education, Inc.
Antiparallel Elongation
 Along one template strand of DNA, the DNA
polymerase synthesizes a leading strand
continuously, moving toward the replication fork
Animation: Leading Strand Rightclick slide / select “Play”
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.
10
Antiparallel Elongation
 To elongate the other new strand, called 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, which
are joined together by DNA ligase
Animation: Lagging Strand Rightclick slide / select “Play”
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.
Figure 16.16a
Figure 16.16b-1
3
5
Template
strand
3
5
Overview
Origin of replication
Leading
strand
Lagging
strand
Lagging strand
2
1
Leading
strand
Overall directions
of replication
Figure 16.16b-2
Figure 16.16b-3
3
3
5
Template
strand
3
5
3
5
RNA primer
for fragment 1
1
5
Template
strand
3
5
3
5
3
RNA primer
for fragment 1
1
3
5
5
3
5
Okazaki
fragment 1
1
3
5
11
Figure 16.16b-4
Figure 16.16b-5
3
3
5
Template
strand
3
5
3
1
3
5
3
RNA primer
for fragment 1
5
5
Template
strand
RNA primer
for fragment 1
5
3
1
3
5
3
5
RNA primer
for fragment 2
5
Okazaki
fragment 1
3
5
1
5
3
3
5
RNA primer
for fragment 2
2
Okazaki
fragment 2
1
Okazaki
fragment 1
1
5
3
2
Okazaki
fragment 2
3
5
3
5
1
3
3
5
5
2
1
3
5
5
3
Figure 16.16b-6
Figure 16.17
3
5
Template
strand
3
5
3
RNA primer
for fragment 1
5
1
Overview
Origin of
replication
Leading
strand
3
Lagging
strand
5
3
5
RNA primer
for fragment 2
Okazaki
fragment 1
5
3
3
5
5
DNA pol III
3
1
3
Overall directions
of replication
Leading strand
2
Okazaki
fragment 2
Leading
strand
Lagging
strand
1
3
3
5
5
Parental
DNA
Primer
5
3
Primase
5
4
2
1
5
3
Lagging strand
DNA pol III
DNA pol I
35
3
3
5
DNA ligase
1 3
2
5
2
1
3
5
Overall direction of replication
Figure 16.17a
Figure 16.17b
Overview
Origin of
replication
Leading
strand
Lagging
strand
Leading
strand
Overall directions
of replication
Leading strand
Leading strand
5
Overview
Origin of
replication
Leading
strand
Lagging
strand
Lagging
strand
Leading
strand
Lagging
strand
Overall directions
of replication
Primer
DNA pol III
3
3
Parental
DNA
Primer
5
3
Primase
5
DNA pol III
4
3
3 5
Lagging strand
DNA pol I
3
2
DNA ligase
1 3
5
12
The DNA Replication Complex
 The proteins that participate in DNA replication
form a large complex, a “DNA replication
machine”
 The DNA replication machine may be stationary
during the replication process
 Recent studies support a model in which DNA
polymerase molecules “reel in” parental DNA and
“extrude” newly made daughter DNA molecules
Animation: DNA Replication Review
Right-click slide / select “Play”
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.
Figure 16.18
DNA pol III
Parental DNA
5
3
3
5
5
5
Connecting
protein
3
Helicase
3
DNA
pol III
 YouTube - DNA Replication Process
 YouTube - DNA Replication (Very realistic
3D animation)
Leading strand
3
5
3
5
Lagging strand
Lagging
strand
template
Replicating the Ends of DNA Molecules
Replication at end of DNA
 Limitations of DNA polymerase create problems
for the linear DNA of eukaryotic chromosomes
 At the end of the DNA strand a small portion
of the strand is not replicated
 The usual replication machinery provides no
way to complete the 5 ends, so repeated rounds
of replication produce shorter DNA molecules
with uneven ends
 So we don’t lose important genetic
information, DNA strands have non-coding
end caps
 This is not a problem for prokaryotes, most of
which have circular chromosomes
 These end caps are called telomeres
© 2011 Pearson Education, Inc.
13
Figure 16.20
Figure 16.20a
5
Leading strand
Lagging strand
Ends of parental
DNA strands
3
Last fragment
5
5
3
Last fragment
Removal of primers and
replacement with DNA
where a 3 end is available
Second round
of replication
Parental strand
5
3
New lagging strand
5
Next-to-last fragment
RNA primer
Lagging strand
5
3
New leading strand
Leading strand
Lagging strand
3
RNA primer
Lagging strand
Parental strand
Ends of parental
DNA strands
Next-to-last fragment
5
3
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Further rounds
of replication
3
Shorter and shorter daughter molecules
Figure 16.20b
Telomeres
5
3
 Eukaryotic chromosomal DNA molecules have
special nucleotide sequences at their ends
called telomeres
Second round
of replication
5
New leading strand
3
New lagging strand
5
 They postpone the erosion of genes near the
ends of DNA molecules
3
 It has been proposed that the shortening of
telomeres is connected to aging
Further rounds
of replication
Shorter and shorter daughter molecules
© 2011 Pearson Education, Inc.
Figure 16.21
Replication at end of DNA
 Telomerase build the telomeres.
 Embryos have high telomerase activity, as
you age you lose this activity.
 Cancer cells have telomerase activity
1 m
14
Fig. 14.24-1
Fig. 14.24-2
Mistakes – repair mechanisms
Causes of Mutations
 Before a cell can divide, it must make a
complete copy of itself
 Random error – sometimes things just go
wrong.
 There are millions of bases that need to be
added to the DNA strands – many chances for
something to go wrong
 Mutagens – chemicals that damage the DNA
and cause mutations in replication
 Enzymes will take out the wrong nucleotide
and replace it with the correct one
Results of Mutations
 A few things can happen if DNA mutates
before the cell replicates:
 Enzymes can repair the damage
 Or – The cell may commit suicide (apoptosis)
 Or – The cell may replicate and the mutation
becomes permanent
 Cigarette smoke
 Sunlight
 Many chemicals (benzene)
Proofreading and Repairing DNA
 In nucleotide excision repair, an endonuclease cuts
out and DNA polymerase replaces damaged
stretches of DNA
© 2011 Pearson Education, Inc.
15
Repair Mechanisms
Fig. 14.25-1
 Photorepair
 UV light can cause pyrimidines dimers to occur.
 Photolyase uses visible light to break dimer
 Nucleotide excision repair
 mismatched pairs are recognized and removed
 an endonuclease cuts out and DNA
polymerase replaces damaged stretches of
DNA then DNA ligase joins the segements
Fig. 14.25-2
Figure 16.19
5
3
3
5
Nuclease
5
3
5
3
DNA
polymerase
3
5
3
5
DNA
ligase
Evolutionary Significance of Altered DNA
Nucleotides
 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
5
3
3
5
Which enzyme unwinds the DNA molecule
and breaks the hydrogen bonds between the
base pairs?
1.
2.
3.
4.
5.
helicase
Topoisomerases
DNA polymerases
Primase
DNA ligase
20%
1
© 2011 Pearson Education, Inc.
20%
20%
2
3
20%
4
20%
5
Copyright © 2009 Pearson Education, Inc.
16
Which enzyme produces breaks in the DNA
molecule to relieve the stress of unwinding,
then they also repair these breaks?
1.
2.
3.
4.
5.
helicase
Topoisomerases
DNA polymerases
Primase
DNA ligase
20%
1
20%
20%
2
3
20%
20%
4
20%
1
Copyright © 2009 Pearson Education, Inc.
3’-TAGC-5’ would pair with
1.
2.
3.
4.
3’-ATCG-5’
3’-CGAT-5’
5’-ATCG-3’
5’-CGAT-3’
helicase
topoisomerases
DNA polymerases
primase
DNA ligase
20%
1
20%
20%
2
3
20%
20%
4
5
Copyright © 2009 Pearson Education, Inc.
Which enzyme adds the RNA bases which starts
the new strands?
helicase
topoisomerases
DNA polymerases
primase
DNA ligase
1.
2.
3.
4.
5.
5
Copyright © 2009 Pearson Education, Inc.
1.
2.
3.
4.
5.
Which enzyme builds the new complementary
strand by adding new nucleotides to the 3’ end
which pair with the old DNA?
20%
20%
2
3
20%
4
Which enzyme joins the Okazaki fragments on the
lagging strand?
20%
1.
2.
3.
4.
5.
helicase
topoisomerases
DNA polymerases
primase
DNA ligase
20%
1
5
20%
20%
2
3
20%
20%
4
5
Copyright © 2009 Pearson Education, Inc.
Chromosome consists of a DNA molecule
packed together with proteins
 The bacterial chromosome is a double-stranded,
circular DNA molecule associated with a small
amount of protein
 Eukaryotic chromosomes have linear DNA
molecules associated with a large amount of protein
 In a bacterium, the DNA is “supercoiled” and found
in a region of the cell called the nucleoid
© 2011 Pearson Education, Inc.
17
Chromosome consists of a DNA molecule
packed together with proteins
 Chromatin, a complex of DNA and protein,
is found in the nucleus of eukaryotic cells
 Chromosomes fit into the nucleus through
an elaborate, multilevel system of packing
Animation: DNA Packing
Right-click slide / select “Play”
© 2011 Pearson Education, Inc.
© 2011 Pearson Education, Inc.
Figure 16.22a
Figure 16.22b
Chromatid
(700 nm)
30-nm fiber
Nucleosome
(10 nm in diameter)
Loops
DNA double helix
(2 nm in diameter)
Scaffold
300-nm fiber
Histones
DNA, the double helix
Histones
Histone
tail
H1
30-nm fiber
Nucleosomes, or “beads on
a string” (10-nm fiber)
Replicated
chromosome
(1,400 nm)
Looped domains
(300-nm fiber)
Chromosome consists of a DNA molecule
packed together with proteins
Metaphase
chromosome
Figure 16.23
 Chromatin undergoes changes in packing during the
cell cycle
 Though interphase chromosomes are not highly
condensed, they still occupy specific restricted
regions in the nucleus
5 m
 At interphase, some chromatin is organized into a
10-nm fiber, but much is compacted into a 30-nm
fiber, through folding and looping
© 2011 Pearson Education, Inc.
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Chromosome consists of a DNA molecule
packed together with proteins
 Most chromatin is loosely packed in the nucleus
during interphase and condenses prior to mitosis
 DNA wrapping around proteins
 Loosely packed chromatin is called euchromatin
 During interphase a few regions of chromatin
(centromeres and telomeres) are highly
condensed into heterochromatin
 Dense packing of the heterochromatin makes it
difficult for the cell to express genetic information
coded in these regions
© 2011 Pearson Education, Inc.
Important Concepts
 Know the vocabulary in this lecture
 Structure of DNA – and their nucleotides
 The four bases, and which are paired together
 Be able to recognize the four base structures
 Know which bases are purines and Pyrimidines
 Type of bonds/linkages
 Be able to draw DNA for me (you can use S
and P for sugar and phosphate, ATCG for
bases, 5’ and 3’)
Important Concepts
 Be able to describe how is DNA replicated
 Semiconservative replication
 Steps
 Complementary pairing
 Direction of building the complementary pair
 The role of helicase, Single-strand binding proteins
Topoisomerases, DNA polymerases, DNA ligase
 Understand how the leading strand is build vs how
the lagging strand is built, know what Okazaki
fragments are,
Important Concepts
 Know what telomers and telomerases are
 What supplies the energy to be used to build the
new strand
 Be able to identify correctly paired bases and
incorrectly paired bases
 Know the repair mechanisms for DNA
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