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CHAPTER 16
MOLECULAR BASIS
OF INHERITANCE
DNA as genetic material?
• Deducted that DNA is the genetic material
• Initially worked by studying bacteria & the
viruses that infected them
• 1928 – Frederick Griffiths – studied
bacterial transformation using
Streptococcus pneumonia
• Used a pathogenic (disease causing) strain &
nonpathogenic strain
• Killed pathogenic strain w/ heat & mixed with living
nonpathogenic strain
• Some of the nonpathogenic transformed to pathogenic
• Oswald-Avery – deducted that the transforming agent
was DNA
Hershey – Chase Experiment
• Used viruses that infect bacteria –
bacteriophage or phage
• T2 is common virus of E. coli –bacteria of
mammal intestines
• Radioactively tagged sulfur in proteins & the
phosphorous in DNA of the T2 phage
• Infected E.Coli with T2 phage
• Radioactive sulfur in supernate – deducted that
protein did not enter bacteria
• Radioactive phosphorous in cells – deducted
that DNA enter cells
T2 Phage
Review of
DNA Structure
• Erwin Chargaff –
studied the nitrogen
bases in the DNA of
many species
• Noticed that amount
of adenine equals
thymine and amount
of cytosine equals
guanine
• Chargaff’s rule
Building a Structural Model of DNA
• Rosalind Franklin & Maurice Wilkins –
used X - ray crystallography to uncover
the double helix of DNA
• Sugar – phosphate backbone on outside
• Watson & Crick – purine paired with a
pyrimidine (purine to purine would make
helix too wide)
• Side groups on each nitrogen base forms
a hydrogen bond between them
Watson & Cricks Hypothesis of
DNA Replication
• Each strand serves as a template for the new
strand
Origin of DNA Replication
1.
2.
3.
Replication begins at
specific sites where the
two parental strands
separate & form
replication bubbles
Bubbles expand
laterally, as DNA
replication proceeds in
both directions
Replication bubbles
fuse & synthesis of
daughter strands is
complete
Antiparallel Elongation
• Enzyme DNA polymerase adds new
nucleotides to the template strands
• Two strands of DNA run antiparallel
(opposite directions)
• DNA polyermase III adds nucleotides in
the 5’ to 3’ direction
• Leading strand – new
strand made in 5’ to 3’
direction
• Lagging strand – strand
produce by nucleotides
being added in the
direction away from the
replication fork ; synthesis
occurs with a series of
segments called Okazaki
fragments
• DNA Ligase – enzyme
that joins the sugar –
phosphate backbones of
Okazaki fragments to
form new DNA strand
Priming DNA Synthesis
• DNA polymerases cannot initiate synthesis
of a polynucleotide (can only add
nucleotides to the 3’ end)
• Primer – initial nucleotide chain; short &
consist of either DNA or RNA ; initiate
DNA replication
• Primase – enzyme that can start a stretch
of RNA from scratch
1. Primase joins RNA nucleotide to primer
2. DNA polymerase III adds DNA
nucleotides to 3’ end of primer
3. Continues adding DNA nucleotides
4. DNA polymerase I replaces the RNA
nucleotide with DNA versions
Proteins For Leading & Lagging
Strands
PROTEIN
FUNCTION
Helicase
Unwinds double helix at
replication fork
Single-strand binding protein Stabilizes single-stranded
DNA until used as a
template
Topiosomerase
Corrects “overwinding”
ahead of replication fork
Protein
Primase
Function for Leading Strand
Synthesizes a single RNA primer
at 5’ end of leading strand
Function for Lagging Strand
Synthesizes an RNA primer at
the 5’ end of each Okazaki
fragment
Elongates each Okazaki
DNA pol III Continuously synthesizes the
leading strand, adding on to primer fragment, adding on to its
primer
DNA pol I
Removes primer from 5’ end of
leading strand & replaces it with
DNA
Removes the primer from the 5’
end of each fragment &
replaces it with DNA, adding on
to 3’ end of adjacent fragment
DNA
Ligase
Joins the 3’ end of DNA that
replaces the primer to rest of
leading strand
Joins Okazaki fragments
Summary of DNA replication
1.
2.
3.
4.
5.
6.
7.
Helicase unwinds double helix
Single-stranded binding proteins stabilize template strands
Leading strand is synthesized continuously in 5’-3’ direction by
DNA pol III
Primase begins synthesis fo RNA primer for 5th Okazaki fragment
DNA pol III completes synthesis of fourth fragment; when it
reaches RNA primer on third fragment it will break off & move to
replication fork, and add DNA nucleotides to 3’ end of fifth
fragment
DNA pol I removes primer from 5’ end of second fragment,
replacing it with DNA nucleotides that it adds one by one to 3’ end
of third fragment. Replacement of last RNA nucleotide with DNA
leaves the sugar-phosphate backbone with a free 3’ end
DNA ligase binds 3’ end of the second fragment to the 5’ end of
the first fragment
1
2
3
7
4
5
6
Proofreading & Repairing DNA
• DNA polymerases proofread nucleotides &
immediately replace any incorrect pairing
• Some mismatched nucleotides evade
proofreading or occur after DNA synthesis
is complete – damaged
• Mismatch repair – cells use special
enzymes to fix incorrect nucleotide pairs
• 130 repairing enzymes identified in
humans to date
Nucleotide Excision Repair
• Thymine dimer – type
of damage caused by
UV radiation
• Buckling interrupts
DNA replication
Replicating Ends of DNA Molecules
• Since only adds to 3’ end, no way to
complete 5’ ends
• Even if Okazaki fragment is started with a
an RNA primer, it can not be replaced with
DNA when removed
• Results in shorter DNA molecules
• Problem exists only in eukaryotes due to
linear DNA
• Prokaryote DNA is circular
• Telomeres – nucleotide sequences at ends of DNA
molecules
• Consist of multiple repetitions of a short sequence
• TTAGGG – human telomere
• Telomeres prevent the erosion of genes near the ends of
DNA molecules
• Somatic cells of older individuals have shorter telomeres
– related to aging?
• In germ cells the erosion of telomeres can lead to loss of
essential genes
• Telomerase – enzyme that catalyzes the lengthening of
telomeres; restores the original length