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Lecture 7 DNA repair
Chapter 10
Problems 2, 4, 6, 8, 10, 12, and 14
Quiz 3 due today at 4:00 PM
10_Figure01.jpg
Types of mutations
Transition mutations
Pyrimidine to __________
Purine to ___________
Transversion mutations
Pyrimidine to __________
Purine to ___________
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Mutations can be permanently fixed if they are not repaired
before the next round of replication
Polymerase errors cause a distortion of the DNA helix
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Mismatch repair of mutations in E. coli
MutS protein recognizes mismatch,
induces a kink in the DNA, and binds ATP
MutS recruits MutL and MutH
MutL activates MutH. MutH nicks DNA
Helicase unwinds strand. An exonuclease
degrades the strand with the mutation
Polymerase III fills in the gap
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MutS complexed with DNA
ATP
kink in DNA
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How does the cell know
which strand contains the
mutated base?
Dam methylase does not
methylate right after replication.
Later A in GATC sequences
are methylated.
MutH nicks the unmethylated
strand of DNA in the mismatch
repair process
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Directionality in mismatch repair
The exonuclease binds the MutH nick and degrades DNA
travelling toward MutS until MutS and the mismatch are found
Exonucleases have a distinct polarity (5’->3’ or 3’->5’) so at least
2 different exonucleases must be used in mismatch repair
Eukaryotic cells lack a MutH homolog
Then how do the mismatch repair enzymes
recognize the newly synthesized strand of DNA?
Nicks present in unligated Okazaki fragments
may play this role following lagging strand DNA
synthesis.
But how nicks get introduced in the leading
strand DNA is not quite clear.
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Hydrolytic damage of DNA
deamination
U base pairs with A
What is the result?
Abasic site (apurinic deoxyribose)
Adenine and guanine also
spontaneously deaminate to
hypoxanthine and xanthine.
depurination
Which type of damage is worst?
deamination
Why didn’t DNA evolve to use U?
10_UnFigure01.jpg
Ames Test to detect mutagens (carcinogens)
Detects reversions
of mutation
A base substitution or frameshift
mutation is introduced in a gene
used to make the amino acid histidine.
Some mutagens need
to be activated in the liver.
So liver extract is commonly
added to the mutagen.
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Base damage by alkylation and oxidation
Alkylation – introduction of methyl or ethyl groups by chemicals
(nitrosamines)
Can form O6-methylguanine
when alkylated
8-oxoguanine (Oxo-G) forms after oxidation. It can basepair with A.
Would this lead to transition or transversion mutation?
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Radiation-induced DNA damage
UV light causes adjacent pyrimidines to covalently bond.
This blocks progression of DNA polymerase.
Gamma and X-ray radiation and some drugs (bleomycin)
induce double stranded breaks in DNA
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Chemicals that cause mutations in DNA
Base analog
of thymidine
What is result?
Intercalating agentsinsert between bases
causing insertions
& deletions in DNA
What type of
interaction with
bases?
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DNA repair systems
10
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Direct DNA repair: Photoreactivation
Visible light is used as the energy to repair thymine dimers
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Direct DNA repair: methyl group removal
Repair of alkylation of O6-methylguanine
A cysteine on the methyltransferase binds the methyl
group on guanine.
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Base excision repair pathway
Example: uracil glycosylase
How does uracil usually get in DNA?
AP site
What type of site is present after glycosylase action?
8 different DNA glycosylases in humans (ex. Oxo-G).
They scan minor groove looking for damaged bases
and then use base flipping to access base for repair.
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Oxo-G glycosylase
Glycosylase protein is gray.
DNA is purple
What is different
about the red base?
10_Figure15.jpg
Oxo-G:A repair
It’s not always too late to repair DNA after replication.
A glycosylase recognizes oxo-G:A and removes the A.
Another glycosylase recognizes G:T basepairs and
removes the T which likely arose from spontaneous
deamination of 5-methylcytosine.
10_Figure16.jpg
Nucleotide excision repair in E. coli
Nucleotide excision repair recognizes
distortions in the double helix.
UvrA+UvrB scan DNA.
UvrA recognizes distortion and leaves.
UvrB melts DNA to form single-stranded bubble
UvrC is recruited and cuts DNA 8 nucleotides
5’ of the legion and 4-5 nucleotides 3’ of
the legion.
Helicase UvrD removes the single strand.
DNA polymerase I and DNA ligase fill the gap.
Nucleotide excision repair (NER) in humans
Similar but more complex than NER in E. Coli.
Mammalian NER uses around 25 proteins.
XPC recognizes distortions (like UvrA in E. coli).
XPA and XPD helicases melt DNA (like UvrB in E. coli).
Single stranded binding protein RPA binds DNA.
5’-cleavage site cut by ERCC1-XPF nuclease and 3’-cleavage
site cut by XPG nuclease (similar to UvrC in E. coli)
24-32 nucleotide long DNA strand is released that is filled in
by a polymerase and sealed by DNA ligase.
Xeroderma pigmentosum disease caused by mutations in XP_
(NER) genes. Patients are susceptiple to cancer from UV light.
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Transcription coupled DNA repair
In humans when transcription of DNA
stalls due to a lesion in DNA, the RNA
polymerase recruits the NER proteins.
The TFIIH complex needed for melting
DNA for transcription contains XPA and
XPD. What is the significance of this?
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Mammalian non-homologous end joining
(NHEJ) pathway to repair double stranded
DNA breaks.
Double stranded breaks (DSB) are
the most toxic of all types of DNA
damage.
Ku70/Ku80 heterodimer binds ends of
DNA and recruits DNA-protein kinase cs.
Artemis, an exo/endonuclease, is
phosphorylated by DNA-PKcs and
processes the DNA ends. Ligase IV
complex attaches the 2 ends together.
NHEJ also used in VDJ recombination
to produce staggering amounts of
different types of antibodies to fight
infections and in Bacillus subtilis
bacterial spores to protect the DNA.
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Translesion DNA synthesis in E. Coli
Erroneous and used as last resort, but used
to replicate through DNA lesions.
Sliding clamp and DNA Pol III fall off DNA.
Pol V contains UmuC
A translesion polymerase (Pol IV or Pol V)
copies across lesion (thymidine dimer).
Translesion polymerase falls off and DNA
Pol III holocomplex resumes replication.
UmuC part of Y family of DNA polymerases.
10_Figure21.jpg
Y family polymerase (left) & high fidelity T7 phage polymerase (right)
translesion
polymerase
normal polymerase
Incoming nucleotides in red and template in blue
What do you notice about the structure around
the active site (yellow arrow)?
10_UnFigure02.jpg
The Y Family of translesion polymerases
Translesion DNA synthesis in E. Coli is induced by SOS DNA damage response.
Adding ubiquitin peptide to sliding clamp at lesion recruits translesion polymerase.
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DNA repair systems
10
Double strand break repair pathway
(homologous recombination)
Uses information from homologous sister
chromosome and will be discussed next
lecture (chapter 11)