Chapter 12

Learning Objective 1
• What evidence was accumulated during the 1940s and early 1950s demonstrating that DNA is the genetic material?

The Mystery of Genes
• Many early geneticists thought genes were proteins
•
•
Proteins are complex and variable
Nucleic acids are simple molecules

Evidence for DNA
•
• DNA (deoxyribonucleic acid)
Transformation experiments
•
DNA of one strain of bacteria can transfer genetic characteristics to related bacteria

Bacteriophage Experiments
• Bacteriophage (virus) infects bacterium
•
• only DNA from virus enters the cell virus reproduces and forms new viral particles from DNA alone

KEY CONCEPTS
• Beginning in the 1920s, evidence began to accumulate that DNA is the hereditary material

Learning Objective 2
• What questions did these classic experiments address?
•
•
•
Griffith’s transformation experiment
Avery’s contribution to Griffith’s work
Hershey –Chase experiments

Griffith’s Transformation Experiment
•
•
Can a genetic trait be transmitted from one bacterial strain to another?
Answer: Yes

Griffith’s Transformation Experiment

Experiment 1 Experiment 2 Experiment 3
Experiment 4
R cells injected
S cells injected
Heat-killed
S cells injected
R cells and heatkilled S cells injected
Mouse lives Mouse dies Mouse lives Mouse dies
Fig. 12-1, p. 261

Animation: Griffith’s Experiment
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Avery’s Experiments
•
•
What molecule is responsible for bacterial transformation?
Answer: DNA

Hershey –Chase Experiments
•
•
Is DNA or protein the genetic material in bacterial viruses (phages)?
Answer: DNA

Hershey –Chase
Experiments

35 S
1
Bacterial viruses grown in 35 S to label protein coat or 32 P to label DNA
32 P
2
Viruses infect bacteria
Fig. 12-2, p. 262

3
Agitate cells in blender
Agitate cells in blender
4
35 S
Separate by centrifugation
Separate by centrifugation
32 P
5
35 S-labeled protein in supernatant
Bacteria in pellet contain 32 Plabeled DNA
Fig. 12-2, p. 262

6
Viral reproduction inside bacterial cells from pellet
7
Cell lysis
5
6
7
32 P
Fig. 12-2, p. 262

Learning Objective 3
• How do nucleotide subunits link to form a single DNA strand?

Watson and Crick
•
• DNA Model
Demonstrated
• how information is stored in molecule’s structure
• how DNA molecules are templates for their own replication

Nucleotides
•
• DNA is a polymer of nucleotides
Each nucleotide subunit contains
•
•
• a nitrogenous base
• purines ( adenine or guanine )
• pyrimidines ( thymine or cytosine ) a pentose sugar ( deoxyribose ) a phosphate group

Forming DNA Chains
•
•
Backbone
•
• alternating sugar and phosphate groups joined by covalent phosphodiester linkages
Phosphate group attaches to
•
•
5 ′ carbon of one deoxyribose
3 ′ carbon of the next deoxyribose

DNA
Nucleotides

Thymine
Adenine Nucleotide
Phosphate group
Cytosine
Phosphodiester linkage
Deoxyribose
(sugar)
Guanine
Fig. 12-3, p. 264

Animation: Subunits of DNA
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KEY CONCEPTS
• The DNA building blocks consist of four nucleotide subunits: T, C, A, and G

Learning Objective 4
• How are the two strands of DNA oriented with respect to each other?

DNA Molecule
• 2 polynucleotide chains
• associated as double helix

DNA Molecule

3.4 nm
Sugar –phosphate backbone
Minor groove
Major groove
= hydrogen
= atoms in base pairs
0.34 nm
2.0 nm
= carbon
= oxygen
= phosphorus
Fig. 12-5, p. 266

Double Helix
•
•
•
Antiparallel
• chains run in opposite directions
5 ′ end
• phosphate attached to 5 ′ deoxyribose carbon
3 ′ end
• hydroxyl attached to 3 ′ deoxyribose carbon

KEY CONCEPTS
•
•
The DNA molecule consists of two strands that wrap around each other to form a double helix
The order of its building blocks stores genetic information

Animation: DNA Close Up
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Learning Objective 5
•
• What are the base-pairing rules for DNA?
How do complementary bases bind to each other?

Base Pairs
•
•
•
Hydrogen bonding
•
• between specific base pairs binds two chains of helix
Adenine ( A ) with thymine ( T )
• forms two hydrogen bonds
Guanine ( G ) with cytosine ( C )
• forms three hydrogen bonds

Base Pairs and Hydrogen Bonds

Fig. 12-6a, p. 267

Adenine Thymine
Deoxyribose
Guanine Cytosine
Deoxyribose
Deoxyribose Deoxyribose
Fig. 12-6b, p. 267

Chargaff’s Rules
•
•
Complementary base pairing
•
• between A and T; G and C therefore A = T; G = C
If base sequence of 1 strand is known
• base sequence of other strand can be predicted

KEY CONCEPTS
•
•
Nucleotide subunits pair, based on precise pairing rules: T pairs with A, and C pairs with G
Hydrogen bonding between base pairs holds two strands of DNA together

Learning Objective 6
• What evidence from Meselson and Stahl’s experiment enabled scientists to differentiate between semiconservative replication of DNA and alternative models?

Models of DNA
Replication

(a) Hypothesis 1: Semiconservative replication
Parental DNA First generation Second generation
Fig. 12-7a, p. 268

(b) Hypothesis 2: Conservative replication
Parental DNA First generation Second generation
Fig. 12-7b, p. 268

(c) Hypothesis 3: Dispersive replication
Parental DNA First generation Second generation
Fig. 12-7c, p. 268

Meselson-Stahl Experiment
•
•
E. coli
•
• grown in medium containing heavy nitrogen
( 15 N) incorporated 15 N into DNA
Transferred from 15 N to 14 N medium
• after one or two generations, DNA density supported semiconservative replication

Meselson-Stahl
Experiment

Bacteria are grown in
15 N (heavy) medium. All
DNA is heavy.
Some cells are transferred to
14 N (light) medium.
Some cells continue to grow in 14 N medium.
First generation Second generation
Cesium chloride
(CsCl)
DNA
DNA is mixed with CsCl solution, placed in an ultracentrifuge, and centrifuged at very high speed for about 48 hours.
High density
Low density
The greater concentration of CsCl at the bottom of the tube is due to sedimentation under centrifigal force.
14 N (light)
DNA
14 N – 15 N hybrid DNA
15 N (heavy)
DNA
DNA molecules move to positions where their density equals that of the CsCl solution.
Fig. 12-8a, p. 269

14 N – 15 N hybrid DNA
14 N (light)
DNA
14 N – 15 N hybrid DNA
15 N (heavy)
DNA
Before transfer to 14 N
One cell generation after transfer to 14 N
Two cell generations after transfer to 14 N
The location of DNA molecules within the centrifuge tube can be determined by UV optics. DNA solutions absorb strongly at 260 nm.
Fig. 12-8b, p. 269

Semiconservative Replication
• Each daughter double helix consists of
•
•
1 original strand from parent molecule
1 new complementary strand

Learning Objective 7
•
• How does DNA replicate?
What are some unique features of the process?

DNA Replication
•
•
•
2 strands of double helix unwind
• each is template for complementary strand
Replication is initiated
•
DNA primase synthesizes RNA primer
DNA strand grows
•
DNA polymerase adds nucleotide subunits

DNA Replication

Nucleotide joined to growing chain by
DNA polymerase
Base
Phosphates released
Fig. 12-10, p. 271

Other Enzymes
•
•
DNA helicases
• open the double helix
Topoisomerases
• prevent tangling and knotting

KEY CONCEPTS
• DNA replication results in two identical double-stranded DNA molecules
• molecular mechanism passes genetic information from one generation to the next

Learning Objective 8
• What makes DNA replication (a) bidirectional and (b) continuous in one strand and discontinuous in the other?

Bidirectional Replication
•
•
Starting at origin of replication
• proceeding in both directions
Eukaryotic chromosome
•
• may have multiple origins of replication may replicate at many points at same time

Bidirectional Replication

3’
5’
DNA polymerase
Origin of replication on DNA molecule
3’
5’
Fig. 12-11a, p. 272

Twist introduced into the helix by unwinding
RNA primer
3’
5’ 3’
DNA helicase
3’
RNA primer
Single-strand binding proteins
DNA polymerase
3’
5’
Direction of replication
Fig. 12-11b, p. 272

3’
5’
3’
5’
3’
5’
3’
5’
Fig. 12-11c, p. 272

DNA Synthesis
•
•
•
Always proceeds in 5 ′ → 3′ direction
Leading strand
• synthesized continuously
Lagging strand
•
•
•
• synthesized discontinuously forms short Okazaki fragments
DNA primase synthesizes RNA primers
DNA ligase links Okazaki fragments

DNA Synthesis

RNA primer
5’
3’
DNA polymerase
3’
5’
Leading strand
DNA helix
3’
5’
Replication fork
Direction of replication
Lagging strand
(first Okazaki fragment)
3’
5’
Fig. 12-12a, p. 273

5’
3’
RNA primers
3’
5’
5’3’
Leading strand
3’
5’
Two Okazaki fragments
5’
3’
Fig. 12-12b, p. 273

5’
3’
DNA ligase
3’
5’
Leading strand
5’
3’
3’
5’
Third Okazaki fragment
5’
3’
Lagging strand
Fig. 12-12c, p. 273

Replication in Bacteria and
Eukaryotes

Template DNA
(light blue)
New DNA (dark blue)
5’
3’
5’
3’
Fig. 12-13a, p. 274

340 nm
Fig. 12-13b, p. 274

Replication
“bubbles”
3’
5’
Replication fork
5’
3’ Single replication bubble formed from two merged bubbles
Fig. 12-13c, p. 274

Animation: Overview of DNA replication and base pairing
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Learning Objective 9
• How do enzymes proofread and repair errors in DNA?

DNA Polymerases
•
•
Proofread each new nucleotide
• against template nucleotide
Find errors in base pairing
•
• remove incorrect nucleotide insert correct one

DNA Mutation

Mutation
Fig. 12-9, p. 270

Mutation
Stepped Art
Fig. 12-9, p. 270

Mismatch Repair
•
•
Enzymes recognize incorrectly paired nucleotides and remove them
DNA polymerases fill in missing nucleotides

Nucleotide Excision Repair
•
•
Repairs DNA lesions
• caused by sun or harmful chemicals
3 enzymes
•
•
• nuclease cuts out damaged DNA
DNA polymerase adds correct nucleotides
DNA ligase closes breaks in sugar –phosphate backbone

Nucleotide Excision Repair

5’
3’
Nuclease enzyme bound to DNA DNA lesion
5’
3’
DNA polymerase
DNA ligase
5’
3’
New DNA
3’
5’
3’
5’
3’
5’
Fig. 12-14, p. 275

Learning Objective 10
•
• What is a telomere ?
What are the possible connections between telomerase and cell aging, and between telomerase and cancer?

Telomeres
•
•
• Eukaryotic chromosome ends
• noncoding, repetitive DNA sequences
Shorten slightly with each cell cycle
Can be extended by telomerase

Replication at Telomeres

5’
3’
DNA replication
5’
3’
5’
3’
RNA primer
+
RNA primer
Removal of primer
3’
5’
3’
5’
3’
5’
5’
3’ 5’
3’
+
3’
5’ 3’
5’
Fig. 12-15a, p. 276

3’
5’
Fig. 12-15b, p. 276

Cell Aging
•
•
May be caused by absence of telomerase activity
Cells lose ability to divide
• after a limited number of cell divisions

Cancer Cells
•
•
Have telomerase
• to maintain telomere length and possibly resist apoptosis
Including human cancers
• breast, lung, colon, prostate gland, pancreas