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Chapter 29/30
DNA Mutation and Repair
Pages 975-978 AND 1009-1012
Learning objectives: Understand the following
• what are the different types of mutation
• how are mutations introduced into DNA
• what are the different types of repair machinery
• how does each repair mechanism function
• how is human disease associated with DNA
repair defects
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Define a Mutation
• A mutation is a stable change in DNA structure
heritable
Base sequence
is changed
• Mutations are classified into several “types”
Molecular Nature of Mutation
(what types of mutations arise)
• Point mutations (base substitutions): one base
substituted for another
• Transition mutations = A to G or C to T (purine to
purine or pyrimidine to pyrimidine)
• Transversion mutations = purine to pyrimidine or vice
versa
• Can arise from mispairing, insertion of base analogs,
or chemical mutagens (nitrous acid, hydroxylamine
and alkylating agents)
Consequences of Point mutations
• A missense mutation: This results in one wrong
codon and one wrong amino acid.
Consequences of Point mutations
A nonsense mutation: If the change in the base
sequence results in a stop codon, the protein would
be terminated at that point in the message.
Consequences of Point mutations
• A sense mutation: This occurs when the change in
the DNA sequence results in a new codon still coding
for the same amino acid.
Other types of mutations
• Insertions and deletions: result in
frameshift mutations (less common)
Insert a G here
DNA Mutation - How?
1) Through mistakes during DNA replication
2) Spontaneous mutations (deamination,
depurination that occur naturally)
3) Induced mutations caused by
environmental agents (chemical mutagens,
UV radiation)
Errors Arising Due to base Mispairing
During DNA Replication (page 975)
1) Tautomerization
2)
3)
Syn vs anti conformation
(flipping around glycosidic bind)
H2O acting as a bridge
Bases can mispair during DNA replication through three (quite rare) mechanisms
that change the H-bonding properties of the base so it is incorporated incorrectly.
Base Analogs may get incorporated during DNA replication
Compounds that chemically resemble a nucleotide base closely enough that
during DNA replication, they can be incorporated into the DNA in place of
the natural base.
= thymine analog
gets incorporated during
DNA replication opposite an A
frequent tautomer change
-now pairs with G
Another base analog
•The base analog,
2-AP pairs with T.
•But 2-AP can
also pair with C
via a singe Hbond.
•Therefore, if 2AP gets in DNA,
then upon
replication a C is
sometimes
inserted.
DNA Mutation - How? - continued
1) Through mistakes during DNA replication
2) Spontaneous mutations (deamination,
depurination that occur naturally)
3) Induced mutations caused by
environmental agents (chemical mutagens,
UV radiation)
Spontaneous DNA Mutation
• Deamination:
- C to U (now pairs with T) (100/cell/day)
- A to Hypoxanthine (now pairs with C)
- G to Xanthine (still pairs with C)
• Depurination/Depyrimidination:
- removes purine base from DNA(104 bases
/cell/day) or pyrimidine bases (less frequent)
• Oxygen radical damage
-breaks sugar rings
Oxidative deamination
The primary amino groups of nucleic acid bases are
somewhat unstable. They can be converted to keto groups
in reactions like the one pictured here:
In a mammalian cell, ~100 uracils are generated per cell per day
in this fashion. Other reactions include conversion of adenine to
hypoxanthine, and guanine to xanthine.
Oxidative deamination
other examples
Spontaneous Base Loss
The glycosyl bond linking DNA
bases with deoxyribose is labile
under physiological conditions.
Within a typical mammalian cell,
several thousand purines and
several hundred pyrimidines are
spontaneously lost per cell per
day.
Loss of a purine or pyrimidine
base creates an
apurinic/apyrimidinic (AP) site
(also called an abasic site)
Oxygen radical damage
DNA Mutation - How?- continued
1) Through mistakes during DNA replication
2) Spontaneous mutations (deamination,
depurination that occur naturally)
3) Induced mutations caused by environmental agents (chemical mutagens, UV
radiation)
Mutation through Exposure to
Environmental Agents
•
Chemical mutagens:
- Nitrous acid (HN02) formed from nitrites (in
preserved meats) reacting with stomach acid,
causes oxidative deamination
- Alkylating agents add methyl or ethyl groups to
bases and change their pairing properties
- Intercalating agents (ethidium bromide, acridine
orange) distort the helix causing frameshift
insertions or deletions.
Agents causing oxidative deamination
Nitrous acid
Nitrosamines
Agents causing alkylation
The first report of mutagenic action of
a chemical was in 1942 by Charlotte
Auerbach, who showed that nitrogen
mustard (component of poisonous
mustard gas used in World Wars I and
II) could cause mutations in cells.
Intercalating Agents
Mutation through Exposure to
Environmental Agents
• Ultraviolet Radiation: classified in terms of
its wavelength
•UV-C (180-290 nm)--"germicidal"--most energetic and lethal, it
is not found in sunlight because it is absorbed by the ozone layer
•UV-B (290-320 nm)--major lethal/mutagenic fraction of sunlight
•UV-A (320 nm--visible)--"near UV"--also has deleterious
effects (primarily because it creates oxygen radicals) but it
produces very few pyrimidine dimers.
Tanning beds have UV-A and UV-B.
•
Ultraviolet Radiation:
- causes dimers to form between adjacent T
bases on a strand of DNA (or between adjacent
T-C bases, hence the term pyrimidine dimer)
- a cyclobutyl ring links the adjacent T’s
- this interferes with the ability of the T’s to
base pair to the opposite strand, and
blocks DNA replication
The most frequent
photoproducts are bonds
formed between adjacent
pyrimidines within one strand
The most frequent are CPDs,
cyclobutane pyrimidine dimers
T-T CPDs are formed most
readily, followed by T-C or C-T;
C-C dimers are least abundant.
T-T CPD with
cyclobutyl ring in blue
CPDs cause a distortion in the
DNA chain structure. The two
adjacent pyrimidines are pulled
closer to each other than in
normal DNA.
DNA Repair
A fundamental difference from RNA, protein,
and lipid
• All these others can be replaced, but DNA must
be preserved
• We have seen how changes get stably
incorporated into DNA
• How do the mutations get repaired??
• E. coli is the model system for understanding
different repair mechanisms
Mechanisms of DNA Repair
• Direct repair - fixes pyrimidine dimers (UV
damage)
• Excision repair:
i) Base Excision Repair (BER) - fixes abnormal
bases (uracil, hypoxanthine, alkylated bases)
ii) Nucleotide Excision Repair (NER) - fixes
large structural changes and helix distortions
(pyrimidine dimers, bulky base adducts)
• Mismatch repair - fixes mismatches
Direct Repair System
Repairs damage without removing the damaged base
Pyrimidine dimers can be repaired by:
DNA photolyase:
•Uses energy from light absorption
•Contains chromophores (light absorbing
agents)
•Action spectrum is blue/near UV light
range
Steps in the repair mechanism:
1) Enzyme recognizes and binds to the
damage
2) Light absorption by chromophore converts
it to an excited state
3) Chromophore donates an electron to the
cyclobutyl dimer
4) Dimer is destabilized and undergoes a
series of electron rearrangements which
result in monomeric pyrimidines
Direct Repair System
Repairs damage without removing the damaged base
Photolyase contains 2 chromophores that absorb light energy. In
all photolyases, one of the chromophores is FADH-, and the other
is either methenyl-tetrahydrofolate (MTHF) or 8-hydroxy-5deazaflavin (8-HDF). MTHF and 8-HDF act as primary light
gatherers (green in the diagram), transferring their energy to
FADH- (yellow). The energy from FADH- is used to split the dimer
Direct Repair System
Repairs damage without removing the damaged base
• CPD photolyases are found in bacteria, fungi, plants
and many vertebrates, but not in placental mammals.
• As we shall see, humans use a different repair pathway
to fix pyrimidine dimers caused by UV light
Excision Repair
• Excision repair:
i) Base Excision Repair (BER) - fixes
abnormal bases (uracil, hypoxanthine,
alkylated bases)
ii) Nucleotide Excision Repair (NER) - fixes
large structural changes and helix
distortions (pyrimidine dimers, bulky base
adducts)
Base Excision Repair
• Abnormal bases are recognized by a set of
DNA glycosylase enzymes, each one
recognizing a specific abnormal base
• For example, uracil glycosylase
recognizes uracil in DNA
• After recognizing the damage the DNA
glycosylase binds to it and initiates a repair
pathway by hydrolyzing off the base
A DNA glycosylase
binds to damaged base
Cleaves N-glycosidic bond
AP endonuclease binds and
cleaves the DNA backbone
Exonuclease removes a
segment of DNA
DNAP fills in the gap
Ligase seals the nick
Note that the AP site
Is identical to that
made by spontaneous
depurination or
depyrimidination.
Steps in Base Excision Repair
1. Removal of the incorrect base by an appropriate
DNA glycosylase to create an AP site
2. Nicking of the damaged DNA strand by AP
endonuclease upstream of the AP site, thus creating
a 3'-OH terminus adjacent to the AP site
3. Excision of the AP site, followed by extension of the
3'-OH terminus by a DNA polymerase
Base Excision Repair is not Enough
• BER can’t deal with all types of damage since
it requires a DNA glycosylase to recognize
each specific damage.
• The huge variety of chemical mutagens
combined with radiation and oxygen radicals
make too many types of damage to each be
recognized by a different DNA glycosylase.
Nucleotide Excision Repair
• Fortunately, a different, more flexible damage
repair mechanism called NER has evolved.
• NER machinery uses a limited number of
proteins to recognize damaged regions in
DNA based on their abnormal structure as
well as on their abnormal chemistry, then
excises and replaces them.
In all organisms, NER has the same steps:
• Damage recognition
• Binding of a protein complex at the damaged site
• Double incision of the damaged strand several
nucleotides away from the damaged site, on both
the 5' and 3' sides
• Removal of the damage-containing fragment from
between the two nicks
• Filling in of the resulting gap by a DNA polymerase
• Ligation
Nucleotide Excision Repair
• Fixes damage causing large distortions in
the helical structure (e.g. UV-induced
pyrimidine dimers, other base adducts)
• In E. coli the NER machinery repairing UV
damage is a protein complex:
UvrA + UvrB + UvrC = ABC excinuclease
• UvrD helicase, DNAP I, and ligase also
participate
Nucleotide Excision Repair in E. coli
• 2 UvrA proteins form
a complex with one
UvrB protein in an
ATP-dependent
reaction
• The complex
recognizes UV
damage by distortion
in the helix
• The UvrA proteins
dissociate from the
complex after ATP
hydrolysis.
• This leaves UvrB
bound across from
the damage
Nucleotide Excision Repair in E. coli
• Now UvrB can recruit UvrC
protein to the complex
• UvrC activates UvrB to
nick the DNA 4 ntds 3’ from
the pyrimidine dimer
• Then UvrB activates UvrC
to nick the DNA 7 ntds 5’
from the pyrimidine dimer
• This leaves a fragment of
DNA containing the
damage that can now be
removed
Nucleotide Excision Repair in E. coli
• A helicase, UvrD,
uses ATP hydrolysis
to power the
unwinding of the
damaged DNA
fragment. This
reomoves UvrC
• The gap in the DNA is
now filled in by
DNAPI or II, reomving
uvrB in the process
• Finally, DNA ligase
seals the nick
NER in Humans
• Critical for repairing UV-induced damage
(because we don’t do direct repair)
• The principal is the same as in bacteria:
i.e. damage is recognized, an excinuclease
makes a gap, DNAP fills the gap
• But the proteins are different (20-30)
• Defects in NER proteins cause genetic
disorders
NER in Humans
Xeroderma pigmentosum (XP)
Genetic disorder with symptoms:
-extreme sensitivity to sunlight (by ~age 2),
and >1000X higher risk of skin cancer
(by ~age 8)
Defect is in repair of UV damage
Gene mapping identified several
repair proteins (called XP proteins)
XP-C and XP-A recognize pyrimidine dimers
XP-B and XP-D have helicase activity
XP-G and XP-F have nuclease activity
NER in Humans
XP-C recognizes pyrimidine dimers
XP-A binds to the pyrimidine dimer
and helps to recruit other proteins
to a complex
XP-B and XP-D are helicases that
separate the DNA strands around
the damage. RPA keeps the strands
apart
XP-G and XP-F are endonucleases
that cut the DNA on either side of
the damage
The cut fragment is removed and
the gap is filled in by DNAP d or e
Real-world biochemistry
In 1997, NASA
developed a Prototype
UV garment for
children with XP,
Porphyria and other
sun Related Disorders
to have a Quality of life
and Freedom. This
NASA UV Protective
Project designed for XP
was completed in 1998
and the UV garments
are being supplied to
children of parents that
have requested them.
Mismatch Repair
• Corrects mismatches after DNA replication
• Increases replication fidelity by 102-103 fold
• Mismatch repair systems scan newlyreplicated DNA duplexes for mismatched
bases
• PROBLEM: which of the 2 strands has the
correct sequence and which has the error?
Mismatch Repair
• Since the parent (template) strand should
have the correct sequence, repair
machinery identifies the parent strand and
copies it to correct the mistake
• Since methylation occurs post-replication,
repair proteins identify methylated strand
as parent, remove mismatched bases on
other strand and replace them
Steps in Mismatch Repair
• MutS recognizes mismatches and binds to
them. Binding of MutL stabilizes the complex.
• The MutS-MutL complex activates MutH,
which locates a nearby methyl group and
nicks the newly synthesized strand opposite
the methyl group.
• A helicase (UvrD) unwinds from the nick in
the direction of the mismatch, and a singlestrand specific exonuclease cuts the unwound
DNA
• the gap is filled in by DNAP III and sealed by
DNA ligase.
Repair of Mismatch Replication Errors
(in E. coli)
1.
3.1. MutS recognizes
mismatches and binds to
them. Binding of MutL
stabilizes the complex.
2.
2. The MutS-MutL
complex activates MutH,
4.
which locates a nearby
methyl group and nicks
the newly synthesized
strand opposite the
methyl group.
Repair of Mismatch Replication Errors
(in E. coli)
1.
3. A helicase (UvrD)
unwinds from the nick in
the direction of the
mismatch, and a singlestrand
specific
2.
exonuclease cuts the
unwound DNA
4. The gap is filled in by
DNAP III and sealed by
DNA ligase.
3.
4.
Continued from the previous slide
Eukaryotic Mismatch Repair
• Detailed mechanism is unclear
• Identification of the parent strand is different
• Human proteins were found similar in
sequence to MutL and MutS
• called hMutL (MLH) and hMutS (MSH)
• Defects in either MLH or MLS result in an
enhanced susceptibility to cancer
(HNPCC=hereditary non-polyposis colon cancer)
We have now finished
Chapters 29 and 30
For next class please read:
Chapter 31
Pages 1014-1023
All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company,
6277
Sea Harbor Drive, Orlando, Florida 32887-6777