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Chapter 16:
Molecular
Basis of
Inheritance
The Race
for the
Double
Helix .
The Search for the Genetic
Material
A. Friedrich Miescher (1868; Swiss)
nuclein
B. Robert Feulgen (1914; German)
DNA staining
C. Frederick Griffith (1928; English)
Streptococcus pneumoniae
Injected mice with a
virulent strain
(smooth because of
a coat)
Streptococcus pneumoniae
Injected mice with a
nonvirulent strain
(rough)
It lacks a capsule; is
harmless
Streptococcus pneumoniae
Boiled the virulent strain.
Heat-killed S cells are
harmless
Streptococcus pneumoniae
Boiled the virulent S
strain.
Combined it with nonvirulent R strain
What do you predict
happened to the mice?
Figure 16.1 Transformation of bacteria
We know this process today as bacterial
transformation.
But what is the transforming factor?
Is it DNA or Protein?
THE SEARCH FOR THE
TRANSFORMING FACTOR
D. Avery, Macleod and McCarty
S strain fractionated
• RNA
• Protein
• Lipid
• Carbohydrate
• DNA
E.
HERSHEY AND CHASE
bacteriophages
Figure 16.2ax Phages
Figure 18.4 The lytic cycle of phage T4
Figure 16.2b The Hershey-Chase experiment
The experiment showed that T2 proteins remained outside the
host cell during infection, while T2 DNA enters the cell
F. Hershey-Chase Experiment
• Results published in 1952
• 1969 Nobel Prize
– Delbruck, Luria and Hershey
F. Other Evidence
1. Amount of DNA doubles prior to
mitosis
2. Diploid chromosomes have twice as
much DNA as haploid sets found in
the gametes of the same organism.
G. Erwin Chargaff 1947
1. Studied the DNA of various
species
2. While the % of A,T,C,G varied
between species, the amount
of A = T, and C = G
3. Chargaff’s Rules
Figure 16.3 The structure of a DNA stand
Figure 5.1 Building models to study the structure and function of macromolecules
Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
Deductions about DNA made from
Franklin’s Photo
1. DNA is a helix with a uniform width of 2
nm.
2. Purine and pyrimidine bases are stacked
.34 nm apart
3. The helix makes one full turn every 3.4
nm along its length.
4. There are ten layers of nucleotide pairs
in each turn of the helix.
Model postulates:
1.The sugar-phosphates alternated on
the exterior of the molecule
2.The nitrogen bases were in the
interior, each one bonded to a sugar.
Model postulates:
3. The two sugar-phosphate backbones of
the helix are antiparallel.
4. A purine (adenine or guanine) must
pair with a pyrimidine (cytosine or
thymine).
5. This base pairing dictates which pairs of
bases can hydrogen bond: A-T, C-G
6. Base pairing rule explains Chargaff’s
rules
Figure 16.5 The double helix
Unnumbered Figure (page 292) Purine and pyridimine
Figure 16.6 Base pairing in DNA
Figure 16.8 Three alternative models of DNA replication
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 2)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 3)
ONE Layers was obtained.
What does this show?
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 4)
After 2nd replication
TWO bands appear.
What does this show?
B. Replication Background
Just how big is your genome?
Your genome is 6B bp (3B X 2 chromosomes)
If printed out the size of your textbook font, this
would fill
1200 Campbell Biology Texts!
B. Replication Background
1. 5M bp with E. coli; 3B bp in humans!
2. Complex
3. Rapid (up to 500 nucleotides/second in
bacteria, 50/sec in human cells)
4. Accurate ( errors only 1/10B)
5. Enzymes: Requires the cooperation of
over a dozen
C. Process of Replication
Origins of Replication: Bacteria have only 1
Replication forks
Eukaryotes may have hundreds or
thousands of origins of replication
Incorporation of a nucleotide into a DNA strand
The nucleotides added are actually triphosphates; the
hydrolysis of the pyrophosphate is the exergonic reax
that drives polymerization.
Figure 16.12 The two strands of DNA are antiparallel
New nucleotides can only be
added at the 3’ end. This
presents a problem!
Figure 16.14 Priming DNA synthesis with RNA
But first . . .
Primers: short segments of RNA polymerized by RNA
primase
Figure 16.14 Priming DNA synthesis with RNA
Then . . .
DNA polymerase III
•13 known active sites
•Self-correction unit
(limits errors to 1/107
bp)
•Can only add new bp to
3’ end
DNA Synthesis
• All synthesis is done from 3’ to 5’ end.
• Proceeds smoothly in one direction =
LEADING STRAND
• Discontinuous in the other direction
=LAGGING STRANDS
• OKAZAKI FRAGMENTS
Synthesis of leading and lagging strands during DNA replication
LIGASE
Is the “glue”
required to
connect the
fragments
A summary of DNA replication
Enzymes of Synthesis
Helicase
Untwists
Topoisomerase
Nicks
SSB
Holds apart
Enzymes of Synthesis
Primase
Makes RNA Primer
Ligase
Connects Okazaki fragments
DNA Polymerase III
Adds new nucleotides 5’ to 3’
DNA Polymerase I
Removes RNA primer; replaces
it with DNA
A--CATALYZES 5’-3’
ADDITON OF NUCLEOTIDES
E- PROOFREADS 3’-5’
B2 DIMER CLAMPS AROUND
DOUBLE HELIX (yellow and blue)
Figure 16.15 The main proteins of DNA replication and their functions
Nobel Prize 1959
Kornberg for DNA Polymerase
Nobel Prize 1962
Watson, Crick and Wilkins
PROOFREADING AND REPAIR
Amnesty Order of Caesar:
EXECUTE NOT, LIBERATE!
However, slightly altered:
EXECUTE, NOT LIBERATE!
PROOFREADING AND REPAIR
DNA is only macromolecule to be repaired
a. Correct nucleotide has higher affinity for
moving polymerase
b. DNA polymerase is a “self-correcting”
enzyme; has a 3’ to 5’ proofreading
exonuclease
PROOFREADING AND REPAIR
Errors in completed molecule: 1/1 billion
c. Mismatch repair enzymes; mutation in
one of these assoc. with colon cancer.
d. Reactive chemicals and UV can
contribute to DNA alterations.
e. There are over 50 130 repair enzymes in
humans!
A. Causes of DNA Damage
Environmental Agents
a. UV Light
1. Thymine or cytosine dimers
2. Distortion interferes with replication and
protein synthesis
Figure 16.17 Nucleotide excision repair of DNA damage
A. Causes of DNA Damage
Environmental Agents
b. Ionizing Radiation: X rays, Atom bomb, 1986
Chernobyl
1. Radiation reacts w/ DNA or water molecules
A. Causes of DNA Damage
Environmental Agents
c. Chemical Agents (Carcinogens)
1. Benzopyrene
2. Cigarette smoke, auto exhaust
3. Dioxin
PROOFREADING AND REPAIR
Nucleotide Excision Repair:
•
DNA Polymerase I (a nuclease) snips out
wrong base, puts in correct nucleotide
•
DNA ligase seals it back
•
Thymine dimers are common with UV damage;
Xeroderma pigmentosum
B. DNA Repair: Enzymatic
Processes
1. Selection of Correct Nucleotide
a. Correct base is most energetically
favorable
b. Error occurs about every
1/100,000 bases
B. DNA Repair: Enzymatic
Processes
2. “Proofreading”
a. Each nucleotide must be
complementary
b. Exonucleases remove
mismatched nucleotides as added
c. Now errors reduced to 1/10M bp
B. DNA Repair: Enzymatic
Processes
3. Mismatch Repair
a. Occurs after synthesis
b. Proteins excise damage
c. Polymerases synthesize new
strands
d. Error rate reduced to 1/10B bp
C. REPAIR ENZYMES
1. Often function after damage has been
done
2. Over 130 known in humans
a. Photolyase: activated by
absorbing visible light; breaks
dimers apart
b. UV repair enzymes: uvrA, B, C
These are nucleases: Remove
damaged sections of nucleotides
C. REPAIR ENZYMES
4. Cancer
Often caused by unrepaired DNA
damage
Ex. Xeroderma pigmentosum
Xeroderma pigmentosum
1. Skin cells defective in excision
repair enzymes
2. Can develop hundreds of skin
cancers
3. “freckled”
4. Internal Cancers
TELOMERES Revisited
1. 1972: James Watson noticed that
the DNA polymerases could not
start at the very tip of a DNA
strand.
2. Analogy: Copy Machine
3. What could you do to be sure the
important information on each
page remains?
Definition/Information
1. TELOMERES: TTAGGG repeats
perhaps 2000 times!
2. In your body, shortening at the
rate of about 31 bp/year
Definition/Information
1. 80 year old person, telomeres are
about 5/8 length at birth
How can this be remedied?
1. Egg and SPERM cells and other
cells in other organisms have
overcome this problem
2. TELOMERASE Enzyme that
catalyzes the addition of lost DNA
by using an RNA template
How can this be remedied?
1. Stopwatch = Telomere
deterioration
2. Germ cells never start the watch.
3. Natural selection has built our
telomeres so they can survive at
most 70-90 years.
4. People from long-lived families
may have longer telomeres
How can this be remedied?
5. Remember the HeLa Cells?
6. Worldwide, weigh more than 400
times her own body weight
7. Oct. 11 is recognized as Henrietta
Lacks Day in Atlanta
8. HeLa cells have excellent
telomerase
Cancer Cells and Telomerase
• Switching on of telomerase genes is an
essential mutation that must occur if a
cancer is to turn malignant
Figure 16.18 The end-replication problem
Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
Figure 16.19b Telomeres and telomerase