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Chapter 16 The Molecular Basis of Inheritance
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA, the substance of inheritance
• Is the most celebrated molecule of our time
• Hereditary information is encoded in the
chemical language of DNA and reproduced in
all the cells of your body
• It is the DNA program that directs the
development of many different types of traits
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DNA is the genetic material
• Early in the 20th century
– The identification of the molecules of
inheritance loomed as a major challenge to
biologists
– Until the 1940’s most scientists believed that
proteins were the genetic material
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The Search for the Genetic Material:
• The role of DNA in heredity
– Was first worked out by studying bacteria and
the viruses that infect them
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Evidence That DNA Can Transform Bacteria
• 1928 - Frederick Griffith was studying
Streptococcus pneumoniae
– A bacterium that causes pneumonia in
mammals
• He worked with two strains of the bacterium
– A pathogenic (one that causes disease) strain
and a nonpathogenic strain
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Evidence that DNA can transform Bacteria
• Griffith found that when he mixed the remains
of heat-killed pathogenic bacteria with
harmless bacteria, some bacteria were
changed into disease-causing bacteria
EXPERIMENT Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they
have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule
and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:
Living S
(control) cells
Living R
Heat-killed
(control) cells (control) S cells
Mixture of heat-killed S cells
and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample.
Figure 16.2
CONCLUSION Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an
unknown, heritable substance from the dead S cells.
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Transformation
• These harmless bacteria had incorporated
external genetic material from the diseasecausing bacteria.
• Griffith called the phenomenon transformation
– Now defined as a change in genotype and
phenotype due to the assimilation of external
DNA by a cell
• He knew a transformation took place, but not
that DNA was the cause of the transformation
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What was the transforming material?
• More than a decade later, Avery, McCarty, and MacLeod
concluded that DNA was the molecule that transformed the
bacteria
– His team purified chemicals from heat-killed
pathogenic cells.
– Basically organic molecules were destroyed and the
team looked at which remaining molecules would allow
for transformation
• For example if RNA, and proteins,lipids and carbs.
were destroyed, transformation would still take
place
• Only when DNA was destroyed did transformation
not occur
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Evidence That Viral DNA Can Program Cells
• Additional evidence for DNA as the genetic
material
– Came from studies of a virus that infects
bacteria
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Evidence That Viral DNA Can Program Cells
• Viruses that infect bacteria, bacteriophages
– Are widely used as tools by researchers in
molecular genetics
Phage
head
Tail
Tail fiber
Figure 16.3
Bacterial
cell
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100 nm
DNA
Hershey - Chase
• 1952 - Alfred Hershey and Martha Chase
– Performed experiments showing that DNA is
the genetic material of a phage known as T2
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• The Hershey and Chase experiment
EXPERIMENT
In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur
and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.
1 Mixed radioactively
labeled phages with
bacteria. The phages
infected the bacterial cells.
Phage
2 Agitated in a blender to 3 Centrifuged the mixture
separate phages outside
so that bacteria formed
the bacteria from the
a pellet at the bottom of
bacterial cells.
the test tube.
Radioactive Empty
protein
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1: Phages were
grown with radioactive
sulfur (35S), which was
incorporated into phage
protein (pink).
Batch 2: Phages were
grown with radioactive
phosphorus (32P), which
was incorporated into
phage DNA (blue).
4 Measured the
radioactivity in
the pellet and
the liquid
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Centrifuge
Radioactivity
(phage DNA)
Pellet
in pellet
RESULTS
Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells.
When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.
Figure 16.4
CONCLUSION
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Chargaff
• 1947 - Erwin Chargaff analyzed the base composition of
DNA from a number of different organisms
•
The ratio of the bases in the DNA was species specific
• Adenines = Thymines
• Guanines = Cytosines
• Became Chargaff’s rules
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DNA Is the Genetic Material
• Prior to the 1950s, it was already known that DNA
– Is a polymer of nucleotides, each consisting of
three components: a nitrogenous base, a
sugar, and a phosphate group
Sugar-phosphate
backbone
5 end
O– 5
O P O CH2
O 1
O– 4 H
H
H
H
2
3
H
O
O P O CH2 O
O–
H
H
H
H
H
O
O P O CH2 O
O–
H
H
H
H
H
Figure 16.5
O 5
O P O CH2
O 1
O– 4 H
H
PhosphateH
H
3 2
OH H
Sugar (deoxyribose)
3 end
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Nitrogenous
bases
CH3
O
H
N
N
H
O
Thymine (T)
H
N
H
N
N
N
N
H
H
Adenine (A)
H H
H
N H
N
N
O
Cytosine (C)
H
N
N
N
O
N H
N H
H
Guanine (G)
DNA nucleotide
Building a Structural Model of DNA:
• Once most biologists were convinced that DNA
was the genetic material
– By the early 1950’s the arrangement of
covalent bonds in a nucleic acid was
established, but the 3-D structure of DNA was
yet to be determined
– The challenge was to determine how the
structure of DNA could account for its role in
inheritance
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Building a Model
• Maurice Wilkins and Rosalind Franklin
– Were using a technique called X-ray
crystallography to study molecular structure
• Rosalind Franklin
– Produced a picture of the DNA molecule using
this technique
Figure 16.6 a, b
(a) Rosalind Franklin
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(b) Franklin’s X-ray diffraction
Photograph of DNA
Watson and Crick’s Double Helix Model of DNA
Paper presented in 1953
Won the Nobel Prize in 1962 along with Wilkins
CrickEnglish
WatsonAmerican
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Building a model of DNA
•
DNA was composed of two antiparallel sugar-phosphate backbones, with the
nitrogenous bases paired in the molecule’s interior
•
The nitrogenous bases
–
Are paired in specific combinations: adenine with thymine, and cytosine
with guanine
G
C
A
T
T
A
1 nm
C
G
C
A
T
G
C
T
A
T
A
A
T
T
A
G
A
Figure 16.7a, c
3.4 nm
G
C
0.34 nm
T
(a) Key features of DNA structure
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(c) Space-filling model
The Structure of DNA
Is like a ladder
The sides of the DNA
molecule are made up of
sugar (deoxyribose) and a
phosphate group.
These are like the
sides of the ladder.
Nitrogen Bases make up the
“rungs” of the ladder.
They are: Thymine,
Adenine, Cytosine
and Guanine.
Deoxyribonucleic Acid
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Building a Model of DNA
•
Watson and Crick reasoned that there must be additional specificity of pairing
–
•
Dictated by the structure of the bases
Each base pair forms a different number of hydrogen bonds
–
Adenine and thymine form two bonds, cytosine and guanine form three
bonds
5 end
O
–O
P
OH
Hydrogen bond
3 end
OH
O
O
A purine must pair with a
pyrimidine
O
–O
P
P
O
G
O
CH2
O
O
C
P
O
O
G
O
O
O
H2C
O
A
(b) Partial chemical structure
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
CH2
O
P
O
O
T
O
OH
3 end
Figure 16.7b
O
C
O
H2C
P
O–
P
O
O
O
CH2
O
O
O
–O
O
O
H2C
–O
A
T
O–
O–
CH2
O
P
O
O
5 end
O–
Base Pairing
H
N
N
N
N
Sugar
O
H
H
CH3
N
N
N
O
Sugar
Thymine (T)
Adenine (A)
H
O
N
N
Sugar
N
H
N
N
N
N
N
Figure 16.8
H
H
Guanine (G)
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H
O
Sugar
Cytosine (C)
Reviewing DNA and RNA
• Campbell Biology
• Chapter 16: The Molecular Basis of Inheritance
–
Activity: DNA and RNA structure
• Go through activity.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Next UP……
DNA Replication
https://www.youtube.com/watch?v=5qSrmeiWsuc
The above video is a simple explanation of what is shown on
the next few slides.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA Replication
• Many proteins work together in DNA replication
• 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• In DNA replication
– The parent molecule unwinds, and two new
daughter strands are built based on basepairing rules
T
A
T
A
T
A
C
G
C
G
C
T
A
T
A
T
A
A
T
A
T
A
T
G
C
G
C
G
C
G
A
T
A
T
A
T
C
G
C
G
C
G
T
A
T
A
T
A
T
A
T
A
T
C
G
C
G
C
A
G
(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
Figure 16.9 a–d
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
The Semiconservative model of DNA replication
Each of the two new daughter molecules will
have one old strand, derived from the parent
molecule, and one newly made strand
First
Second
Parent cell replication replication
(a) Conservative
model. The two
parental strands
reassociate
after acting as
templates for
new strands,
thus restoring
the parental
double helix.
M. Meselson and F. Stahl tested
these models using E. coli
Figure 16.10 a–c
(b) Semiconservative
model. The two
strands of the
parental molecule
separate,
and each functions
as a template
for synthesis of
a new, complementary strand.
(c) Dispersive
model. Each
strand of both
daughter molecules contains
a mixture of
old and newly
synthesized
DNA.
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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
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Getting Started: Origins of Replication
• The replication of a DNA molecule
– Begins at special sites called origins of
replication, where the two strands are
separated
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• A eukaryotic chromosome
– May have hundreds or even thousands of
replication origins
Origin of replication
1 Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
Bubble
Parental (template) strand
Daughter (new) strand
0.25 µm
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
DNA molecule of each chromosome.
Figure 16.12 a, b
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(b) In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
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
5 end
Sugar
Template strand
3 end
A
Base
Phosphate
T
A
T
C
G
C
G
G
C
G
C
A
T
A
OH
Pyrophosphate 3 end
P
OH
Figure 16.13
3 end
5 end
Nucleoside
triphosphate
C
P
C
2 P
5 end
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5 end
DNA Replication
• 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
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DNA Replication
• 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
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• Synthesis of leading and lagging strands during
DNA replication
1 DNA pol Ill elongates
DNA strands only in the
5
3 direction. 3
5
Parental DNA
5
3
Okazaki
fragments
2
1
3
5
DNA pol III
2 One new strand, the leading strand,
can elongate continuously 5
3
as the replication fork progresses.
3 The other new strand, the
lagging strand must grow in an overall
3
5 direction by addition of short
segments, Okazaki fragments, that grow
5
3 (numbered here in the order
they were made).
Template
strand
3
Leading strand
Lagging strand
2
Template
strand
Figure 16.14
1
DNA ligase
Overall direction of replication
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4 DNA ligase joins Okazaki
fragments by forming a bond between
their free ends. This results in a
continuous strand.
Priming DNA Synthesis
• DNA polymerases cannot initiate the synthesis
of a DNA strand
– They can only add nucleotides to an existing
chain that is attached to a template strand.
• An enzyme called primase joins about 10 RNA
nucleotides to form the primer needed to start
the chain
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• 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
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1
Primase joins RNA nucleotides
into a primer.
3
5
5
3
Template
strand
RNA primer
3
5
3
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
2
5
3
1
After reaching the next
RNA primer (not shown),
DNA pol III falls off.
Okazaki
fragment
3
3
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
5
3
2
5
1
DNA pol 1 replaces the
RNA with DNA, adding to
the 3 end of fragment 2.
5
3
6
5
1
DNA ligase forms a bond
between the newest DNA
and the adjacent DNA of
fragment 1.
5
3
Figure 16.15
3
2
7
The lagging strand
in this region is now
complete.
3
2
1
Overall direction of replication
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5
Other Proteins That Assist DNA Replication
• Helicase, topoisomerase, single-strand binding
protein
– Are all proteins that assist DNA replication
Table 16.1
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DNA Replication
• A summary of DNA replication
Overall direction of replication
1 Helicase unwinds the
parental double helix.
2 Molecules of singlestrand binding protein
stabilize the unwound
template strands.
3 The leading strand is
synthesized continuously in the
5 3 direction by DNA pol III.
DNA pol III
Lagging
Leading
strand Origin of replication strand
Lagging
strand
OVERVIEW
Leading
strand
Leading
strand
5
3
Parental DNA
4 Primase begins synthesis
of RNA primer for fifth
Okazaki fragment.
5 DNA pol III is completing synthesis of
the fourth fragment, when it reaches the
RNA primer on the third fragment, it will
dissociate, move to the replication fork,
and add DNA nucleotides to the 3 end
of the fifth fragment primer.
Replication fork
Primase
DNA pol III
Primer
4
DNA ligase
DNA pol I
Lagging
strand
3
2
6 DNA pol I removes the primer from the 5 end
of the second fragment, replacing it with DNA
nucleotides that it adds one by one to the 3 end
of the third fragment. The replacement of the
last RNA nucleotide with DNA leaves the sugarphosphate backbone with a free 3 end.
Figure 16.16
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1
3
5
7 DNA ligase bonds
the 3 end of the
second fragment to
the 5 end of the first
fragment.
Proofreading and Repairing DNA
• DNA polymerases proofread newly made DNA
– Replacing any incorrect nucleotides
• In mismatch repair of DNA
– Repair enzymes correct errors in base pairing
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Proofreading DNA
• In nucleotide excision repair
– Enzymes cut out and replace damaged
stretches of DNA
1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
DNA
polymerase
3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
Figure 16.17
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4 DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
Replicating the Ends of DNA Molecules
• The ends of eukaryotic chromosomal DNA
– Get shorter with each round of replication
5
End of parental
DNA strands
Leading strand
Lagging strand
3
Last fragment
Previous fragment
RNA primer
5
Lagging strand
Primer removed but3
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
3
Removal of primers and
replacement with DNA
where a 3 end is available
5
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Figure 16.18
Further rounds
of replication
Shorter and shorter
daughter molecules
http://www.youtube.com/watch?v=AJNoTmWsE0s
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Telomeres
• Eukaryotic chromosomal DNA molecules
– Have at their ends nucleotide sequences,
called telomeres, that postpone the erosion of
genes near the ends of DNA molecules
Figure 16.19
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1 µm
Telomeres
• If the chromosomes of germ cells became
shorter in every cell cycle
– Essential genes would eventually be missing
from the gametes they produce
• An enzyme called telomerase
– Catalyzes the lengthening of telomeres in
germ cells
http://www4.utsouthwestern.edu/cellbio/shay-wright/intro/facts/sw_facts.html#
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