1. DNA and the Gene: Synthesis and Repair
DNA and the Gene: Synthesis and Repair

2. Chapter 15 Opening Roadmap.
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3. What Are Genes Made Of?
Researchers knew that chromosomes were comprised of DNA and protein
Are genes made of DNA or protein?
Most biologists thought genes were made of proteins
They are relatively more complex
They are more variable
DNA is comprised of only four different nucleotides

4. The Hershey–Chase Experiment
Hershey and Chase studied how the T2 virus infects the bacterium Escherichia coli
T2 infection of E. coli begins when
The virus injects its genes into the cell
These genes direct production of new virus particles
The virus’s protein coat, or capsid, is left behind on the outside of the cell
Does protein or DNA enter the cell?

5. (b) The virus’s capsid stays outside the cell.
Figure 15.1
(a)
Virus capsid
Virus genes
Host cell
1. Start of infection.
2. Production of
new virus
particles.
3. End of infection.
(b) The virus’s capsid stays outside the cell.
Figure 15.1 Viruses Inject Genes into Bacterial Cells and Leave a Capsid Behind.
Virus capsid
Virus genes
100 nm
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6. The Hershey–Chase Experiment
Hershey and Chase grew the virus in the presence of one of two radioactive isotopes:
32P, which is incorporated into DNA, or
35S, which is incorporated into proteins
Labeled viruses were used to infect E. coli cells
Only the radioactive DNA was found inside the cells
Therefore, genes must be composed of DNA

7. Figure 15.2
Do viral genes consist of DNA or protein?
Viral genes consist of DNA.
Viral genes consist of protein.
1. Label viruses.
Viral DNA is
radioactive.
Viral protein
is radioactive.
2. Infect bacteria.
E. coli
E. coli
Viral
capsids
outside
3. Agitate cultures.
Genes
inside
4. Centrifuge solutions.
Viral capsids
in solution
Figure 15.2 Experimental Evidence Showed that DNA Is the Hereditary Material.
Viral genes in
cells in pellet
Radioactive DNA will be
located within pellet.
Radioactive protein will be
located within pellet.
Radioactive
protein is in
solution
Radioactive
DNA is in
pellet
DNA
Viral genes consist of DNA.
Protein
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8. The Secondary Structure of DNA
The primary structure of each strand of DNA has
A backbone made up of the sugar and phosphate groups of deoxyribonucleotides
A series of nitrogen-containing bases that project from the backbone
DNA has directionality
The 3′ end has an exposed hydroxyl group attached to the 3′ carbon of deoxyribose
The 5′ end has an exposed phosphate group attached to a 5′ carbon

9. (a) Structure of a deoxyribonucleotide (b) Primary structure of DNA
Figure 15.3
(a) Structure of a deoxyribonucleotide
(b) Primary structure of DNA
5′ end of strand
Phosphate group
attached to
5′ carbon
of the sugar
Bases project
from the
backbone
Could be
adenine (A),
thymine (T),
guanine (G),
cytosine (C)
Sugar–phosphate
backbone of
DNA strand
Hydroxyl (OH)
group on 3′ carbon
of the sugar
Phosphodiester
bond links
deoxyribonucleotides
Figure 15.3 DNA’s Primary Structure Has a Sugar-Phosphate Backbone.
3′ end of strand
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10. The Secondary Structure of DNA
Watson and Crick proposed that
Two DNA strands line up in the opposite direction to each other in antiparallel fashion
The antiparallel strands twist to form a double helix
The secondary structure is stabilized by complementary base pairing
Adenine (A) hydrogen-bonds with thymine (T)
Guanine (G) hydrogen-bonds with cytosine (C)

11. (a) Complementary base pairing (b) The double helix
Figure 15.4
(a) Complementary base pairing
(b) The double helix 3′ 5′ 5′ 3′
Sugar–
phosphate
backbone
of DNA
Complementary
base pairs held
together by
hydrogen
bonding
Figure 15.4 DNA’s Secondary Structure Is the Double Helix.
Antiparallel strands
(their 5′ → 3′
polarities run in
opposite
directions) 5′ 5′ 3′ 3′
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12. The Secondary Structure of DNA
Because of complementary base pairing
Existing strands of DNA can serve as a template (pattern) for the production of new strands
Three hypotheses for how parental strands and daughter strands act during replication:
Semiconservative replication
Conservative replication
Dispersive replication

13. Testing Early Hypotheses about DNA Synthesis
Semiconservative replication—Parental strands separate, and each is a template for a new strand
Each daughter has one old and one new strand
Conservative replication—The parental molecule serves as a template for an entirely new molecule
One daughter has both old strands; the other has both new strands
Dispersive replication—The parent molecule is cut into sections
Each daughter has old and new DNA interspersed

14. The Meselson–Stahl Experiment
Meselson and Stahl grew E. coli in the presence of “heavy” nitrogen (15N) to label the bacteria’s DNA
Bacteria copy their entire complement of DNA, or genome, before every cell division
Then they moved the bacteria to a normal 14N-containing medium
Allowed the cells to divide once
Separated the DNA by density
The results supported semiconservative replication

15. Figure 15.5
Is replication semiconservative, conservative, or dispersive?
Replication is semiconservative.
Replication is conservative.
Replication is dispersive.
Generation 0
DNA sample
Generation 1
DNA sample
Generation 2
DNA sample
15N
14N
14N
Cell transfer
1. Grow E. coli cells in
medium with 15N as sole
source of nitrogen for many
generations. Collect
sample and purify DNA.
2. Transfer cells to medium
containing 14N. After cells
divide once, collect sample
and purify DNA.
3. After cells have divided
a second time in 14N
medium, collect sample
and purify DNA.
4. Centrifuge the three samples
separately. Compare the locations
of the DNA bands in each sample
to determine density.
Semiconservative replication
Conservative replication
Dispersive replication
Generation 0
15N
15N
15N
Generation 1
Hybrid
Hybrid
15N
14N
Hybrid
Hybrid
Generation 2
Figure 15.5 The Meselson–Stahl Experiment Settled a Key Question about Replication.
Hybrid
14N
Hybrid
14N
15N
14N
Hybrid
After 2 generations:
1/2 low-density DNA (14N)
1/2 intermediate-density DNA (hybrid)
After 2 generations:
1/4 high-density DNA (15N)
3/4 low-density DNA (14N)
After 2 generations:
All intermediate-density DNA
(hybrid)
Top of centrifuge
tube (lower density)
After 2 generations:
1/2 low-density DNA
1/2 intermediate-density DNA
14N
Hybrid
Bottom of centrifuge
tube (higher density)
15N 1 2
Generation
Data from generation 1 conflict with conservative replication hypothesis. Data from generation 2 conflict with dispersive
replication hypothesis. Replication is semiconservative.
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16. A Model for DNA Synthesis
The enzyme that catalyzes DNA synthesis is called DNA polymerase
There are several types of DNA polymerases
They can work only in one direction
Can add deoxyribonucleotides only to the 3′ end of a growing DNA chain
Therefore, DNA synthesis always proceeds in the 5′ → 3′ direction

17. A Model for DNA Synthesis
DNA polymerization should be endergonic
The monomers are deoxyribonucleoside triphosphates (dNTPs)
Have high potential energy because of their three closely packed phosphate groups
They have enough potential energy to make the formation of phosphodiester bonds exergonic

18. Figure 15.6 DNA Synthesis Proceeds in Only One Direction.
Parental strand
Parental strand
3′ end
3′ end
Daughter strand
Daughter strand
5′ end
5′ end
Phosphodiester
bond
3′ end
Figure 15.6 DNA Synthesis Proceeds in Only One Direction.
5′ end
5′ end
Synthesis reaction
3′ end
dNTP
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19. Where Does Replication Start?
A replication bubble forms when DNA is being synthesized
Forms at a specific sequence called the origin of replication
Bacteria have one and form one replication bubble
Eukaryotic cells have many on each chromosome
Each replication bubble has two replication forks
Because synthesis is bidirectional
Replication bubbles grow in two directions

20. (a) DNA being replicated
Figure 15.7
(a) DNA being replicated
(b) Bacterial chromosomes have a single origin of replication.
Old DNA
New DNA
Replication
proceeds 5′ → 3′ in
both directions
Origin of
replication
0.25 μm
(c) Eukaryotic chromosomes have multiple origins of replication.
Replication
fork
Figure 15.7 DNA Replication Forks Move in Two Directions from an Origin of Replication.
Replication
bubble
Old DNA
New DNA
Replication proceeds 5′ → 3′ in both
directions from each starting point
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21. How Is the Helix Opened and Stabilized?
Several proteins are responsible for opening and stabilizing the double helix
DNA helicase breaks hydrogen bonds between the two DNA strands to separate them
Single-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closing
Unwinding the DNA helix creates tension farther down the helix
Topoisomerase cuts and rejoins the DNA to relieve this tension

22. How Is the Leading Strand Synthesized?
DNA polymerase has two parts:
A sliding clamp that forms a ring around the DNA
A part that grips the DNA strand
DNA polymerase
Works only in the 5′ → 3′ direction on a single-stranded template
Requires a 3′ end to extend from

23. How Is the Leading Strand Synthesized?
The 3′ end for DNA polymerase to add to is supplied by a primer
An RNA strand about a dozen nucleotides long
Base-pairs with the DNA template strand
Provides a 3′ hydroxyl (OH) group that can combine with a dNTP to form a phosphodiester bond

24. How Is the Leading Strand Synthesized?
Primers are made by an enzyme called primase
A type of RNA polymerase
Does not require a free 3′ end to begin synthesis
DNA polymerase adds dNTPs to the primer’s 3′ end
Since DNA strands are antiparallel, the synthesis process differs for each strand
The strand that is synthesized toward the replication fork is the leading strand, or continuous strand
It is synthesized continuously in the 5′ → 3′ direction

25. Primase synthesizes RNA primer
Figure 15.8
Primase synthesizes RNA primer
Topoisomerase relieves twisting forces
1. DNA is opened,
unwound, and primed.
Helicase opens double helix
Single-strand DNA–binding proteins (SSBPs) stabilize single strands
Sliding clamp holds DNA polymerase in place
DNA polymerase synthesizes leading strand in 5′ → 3′ direction
RNA primer
2. Synthesis of
leading strand begins.
Figure 15.8 Leading-Strand Synthesis.
Leading strand
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26. How Is the Lagging Strand Synthesized?
The strand synthesized away from the replication fork is the lagging strand or discontinuous strand
It occurs because DNA synthesis must proceed in the 5′ → 3′ direction
The discontinuous replication hypothesis
Primase synthesizes new RNA primers on the lagging strand as the replication fork opens
DNA polymerase synthesizes short fragments of DNA along the lagging strand
Fragments are then linked into a continuous strand

27. Leading strand synthesized 5′ → 3′ DNA unwinds Time 1 Lagging strand
Figure 15.9
Leading strand
synthesized 5′ → 3′
DNA unwinds
Time 1
Lagging strand
synthesized 5′→ 3′
Figure 15.9 The Lagging Strand Is Synthesized Away from the Replication Fork.
Time 2
Region of single-
stranded DNA
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28. The Discovery of Okazaki Fragments
This hypothesis was confirmed by Okazaki
The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments
DNA polymerase I removes the RNA primers and replaces them with DNA
The enzyme DNA ligase joins the Okazaki fragments

29. Figure 15.10 Lagging-Strand Synthesis.
The leading strands are faded out
to help you focus on synthesis
of the lagging strand
RNA
primer
1. Primer added.
Topoisomerase
SSBPs
Helicase
Primase
Okazaki fragment
2. First fragment synthesized.
Sliding clamp
DNA polymerase III
2nd Okazaki fragment
1st Okazaki fragment
3. Second fragment synthesized.
Figure Lagging-Strand Synthesis.
DNA polymerase I
4. Primer replaced.
DNA ligase
5. Gap closed.
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30. Table 15.1 Proteins Required for DNA Synthesis in Bacteria.
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31. The Discovery of Okazaki Fragments
The replisome
Contains the enzymes responsible for DNA synthesis around the replication fork
Joined into one large, multi-enzyme machine

32. Sliding clamp DNA polymerase III Topoisomerase Leading strand Helicase
Figure 15.11
Sliding clamp
DNA polymerase III
Topoisomerase
Leading strand
Helicase
Primase
Figure The Replisome Is a DNA-Synthesizing Machine.
Lagging strand
SSBPs
DNA ligase
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33. Web Activity: DNA Synthesis

34. Replicating the Ends of Linear Chromosomes
Replication of telomeres (ends of linear chromosomes) can be problematic
The leading strand is synthesized all the way to the end
On the lagging strand, primase adds an RNA primer close to the end of the chromosome
The final Okazaki fragment is made and the primer is removed
DNA polymerase cannot add to the end with no primer

35. The End Replication Problem
A single-stranded DNA is left at the end of the lagging strand
The single-stranded DNA is eventually degraded
This would shorten the chromosome by 50 to 100 nucleotides each time replication occurs
Over time, linear chromosomes would vanish
Telomeres do not contain genes
Consist of short, repeating stretches of bases

36. Figure 15.12 Chromosomes Shorten during Normal DNA Replication.
DNA polymerase
End of chromosome
Leading strand
Sliding clamp
1. DNA unwinding
completed.
Lagging strand
Helicase
2. Leading strand
completed.
RNA primer
Primase
3. Lagging strand
nears completion.
Figure Chromosomes Shorten during Normal DNA Replication.
DNA polymerase
Last Okazaki fragment
4. Lagging strand
too short.
No primer for DNA
polymerase; unreplicated end
is eventually lost, shortening
chromosome
Unreplicated end
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37. Telomerase Solves the End Replication Problem
The enzyme telomerase replicates telomeres, using an RNA template that it carries
The 3′ end of the lagging strand forms a single-stranded “overhang”
Telomerase binds to the overhang and uses the RNA that it carries as a template for DNA synthesis
Telomerase continues to move down the new strand, adding more short DNA sequences to the end
Once the overhang is long enough, normal DNA synthesis can occur

38. Missing DNA on lagging strand
Figure 15.13
Missing DNA on
lagging strand
1. End is unreplicated.
Telomerase with its
own RNA template
2. Telomerase extends
unreplicated end.
3. Telomerase repeats activity.
Figure Telomerase Prevents Shortening of Telomeres during Replication.
RNA primer
4. Extended single-strand DNA
acts as template.
DNA polymerase
Sliding clamp
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39. Effect of Telomere Length on Cell Division
Somatic cells normally lack telomerase
Chromosomes of somatic cells progressively shorten
This occurs as the individual ages
Greider proposed that the number of cell divisions is limited by the initial length of a cell’s telomeres
Once chromosome are shortened to a threshold length, further divisions are shut down
In culture, telomere length correlates to number of cell divisions

40. Number of cell divisions 26–50 0–25
Figure 15.14
Donor age
>75
51–75
Number of cell divisions
26–50
0–25
Cells with initially
longer telomeres
divided more
times than
cells with
initially shorter
telomeres,
regardless of
donor age
Figure Telomere Length Predicts the Number of Divisions before Cells Stop Dividing.
Initial telomere length (base pairs)
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41. Effect of Telomere Length on Cell Division
Adding telomerase to cells in culture allows them to continue dividing longer
Most cancer cells have active telomerase
May allow unlimited division of cancer cells
Could inhibiting telomerase slow or stop cancer?

42. Correcting Mistakes in DNA Synthesis
DNA replication is very accurate
Error rate is about one mistake per billion bases
DNA polymerase matches bases with high accuracy
Correct bases are the most energetically favorable
Correct base pairs have a distinct shape
Inserts an incorrect base about once every 100,000 bases
Repair enzymes remove defective bases and replace them with the correct one

43. DNA Polymerase Proofreads
DNA polymerase can proofread its work
Mismatched bases have a distinct shape
DNA polymerase will add a nucleotide only if the previous base pair is correct
If the enzyme finds a mismatch
Its ε (epsilon) subunit removes the mismatched base
This proofreading process reduces the error rate to about one mistake in 10 million base pairs

44. (a) DNA polymerase adds a mismatched deoxyribonucleotide ...
Figure 15.15
(a) DNA polymerase adds a mismatched deoxyribonucleotide ...
(b) ... but detects the mistake and corrects it.
Figure DNA Polymerase Can Proofread.
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45. Mismatch Repair
DNA polymerase sometimes leaves a mismatched pair behind
Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete
Mismatch repair enzymes
Recognize the mismatched pair
Remove a section of the newly synthesized strand that contains the incorrect base
Fill in the correct bases

46. Repairing Damaged DNA
DNA can be damaged by sunlight, X-rays, and many chemicals
Organisms have DNA damage-repair systems
For example, UV light and some chemicals can cause thymine dimers to form
These dimers produce a kink in the DNA strand
This blocks DNA replication

47. adjacent thymine bases
Figure 15.16
UV light
Kink
Figure UV Light Damages DNA.
Thymine
dimer
DNA strand with
adjacent thymine bases
Damaged DNA strand
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48. Repairing Damaged DNA
The nucleotide excision repair system recognizes such types of damage
A protein complex recognizes the kink
Removes the damaged single-stranded DNA
Uses the intact strand as a template for new DNA
DNA ligase links the repaired strand to the original undamaged DNA

49. 1. Error detection. 2. Nucleotide excision. 3. Nucleotide replacement.
Figure 15.17
Damaged bases
Cut
Cut
1. Error detection.
2. Nucleotide
excision.
3. Nucleotide
replacement.
Figure Nucleotide Excision Repair Removes and Replaces Defective Bases.
Repaired damage
4. Nucleotide
linkage.
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50. Xeroderma Pigmentosum: A Case Study
Xeroderma pigmentosum (XP)
A rare autosomal recessive disease in humans
Causes extreme sensitivity to UV light
Increases chance of skin cancer by 1000−2000 times
XP is caused by mutations in nucleotide excision repair systems
The cells of people with XP cannot repair DNA damaged by ultraviolet radiation
Can result from mutations in any of eight genes

51. Unaffected cells XP cells Amount of radioactive nucleotide
Figure 15.18
Amount of radioactive nucleotide
incorporated (counts per minute)
Unaffected
cells
Figure DNA Damage from UV Light IS Not Repaired Properly in Individuals with XP.
XP cells
Dose of UV light (ergs/mm2)
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52. Xeroderma Pigmentosum: A Case Study
Defects in the genes required for DNA repair are frequently associated with cancer
If mutations in the genes involved in the cell cycle go unrepaired
The cell may begin to grow in an uncontrolled manner
This growth can result in the formation of a tumor
If the overall mutation rate in a cell is elevated
Because of defects in DNA repair genes
Then the mutations that trigger cancer become more likely