DNA damage and repair

1
DNA damage and repair

• DNA methylation
• Can also lead to mutations
• DNA repair
• Types of DNA damage
• Types of repair
• Direct repair
• Nucleotide excision repair
• base excision repair
• Postreplication repair
• Mismatch repair

2
Deamination of methylated cytosine results in
base pair changes
- mismatches can also arise from the deamination
of 5-methyl-cytosine - this is a modification
commonly found in eukaryotes - results in
transition mutation of CG ? TA
C ? T
T
A
3
H
H
O
N
H
N
N
T
C
N
O
N
O
5-methyl cytosine
thymine
H2O - NH3
H
H
O
N
H
N
U
N
C
N
O
N
O
uracil
5-methyl cytosine
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DNA Mutation RepairDNA Damage

• Some common types of DNA Damage
• I) Ultraviolet Light
• - major product is thymine dimers (260 nm light)
• ---GCTATTCACGA---
• ---CGATAAGTGCT---
• Blocks replication transcription because helix
  distortion blocks polymerization past this site
• Formed from two adjacent thymine residues joined
  by cyclobutane rings involving carbons 5 and 6
• Survival of UV irradiation direct correlated to
  ability to remove thymine dimers from DNA

5
Thymine dimers major cause of UV-induced
mutations
These can be corrected by a process termed
photo-reactivation Another dimer called a 6-4
dimer is now know to be the major cause of UV
induced mutations
6
Types of Repair

• Direct Repair
• Damaged bases not removed but repaired on site
• Photo-reactivation (DNA photolyase)
• Binds DNA specifically at site of pyrimidine
  dimers
• In the presence of visible light, the binds
  linking the pyrimidine rings are broken and the
  enzyme dissociates
• Process is catalyzed in a process similar to
  photosynthesis- harvesting energy from light
• Human cells do not contain photolyase

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Direct repair

• Alkylation of DNA Bases
• Can block DNA replication because of modified
  bases that are formed
• Sometime used in chemotherapy to block cell
  division
• Usually purines are altered- spectrum of products
  varies
• Most highly mutagenic of these products
• Guanine ? O6 alkyl guanine

CH3
O
Highly mutagenic- base-pairs with T instead of C
N
N
?
N
H2N
N
sugar
guanine
O6-alkyl guanine
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Mispairing of O6-methylguanine with
thymine Crates GC to AT transition
G C
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The other bases and the phosphate group can also
be alkylated
Some Alkylating Agents
Repair of this damage Involves an unusual
enzyme O6-alkylguanine alkyltransferase Which
transfers a methyl or ethyl group from the
O6-methylguanine or O6-ethylguanine residue to a
cysteine within the protein The protein can only
function once- it gets turned over by the cell-
not really an enzyme
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Types of Repair Nucleotide Excision Repair
Excision repair of thymine dimers by E. coli
UvrABC exonucleases - a complex of A and B
proteins track along DNA until it reaches a
thymine dimer or other damaged site, where it
halts and forces the DNA to bend. UvrA then
dissociates, allowing UvrC to bind to B. The BC
complex cuts both sides of the dimer. - helicase,
polymerase and ligase remove the damaged DNA to
replace it with new DNA Enzyme not typical
because it cuts at 2 sites- termed an
excinuclease for its role in excision repair
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Base excision repair (BER)

• Also removes one or more nucleotides from site of
  damage
• Initiates with cleavage of glycosidic bond
  between damaged base and deoxyribose
• Also functions in removal of thymine dimers
• Endonuclease V
• Has two activities
• Glycosylase- cleaves between thymine on 5 side
  of dimer and its deoxyribose
• AP endonuclease- recognizes apyrimidinic (AP)
  site- consists of deoxy ribose without an
  associated pyrimidine base, cleaves on the 5
  side
• Next, deoxyribo-phosphodiesterase cleaves 3 to
  AP site
• Finally, nick translation by DNA pol I removes
  damaged base and DNA ligase closes resultant nick
• Patch may be 1-2 bases long or several bases

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Repair of oxidative damage by BER

• An example of oxidative damage, conversion of
  adenine to 8-oxoguanine
• Highly mutagenic because it can base pair with
  adenine
• Repair can occur through action of mutT
• Cleaves base before DNA incorporation
• Repair can occur through action of mutM
• Removes 8-oxoguanine from DNA
• Repair can occur through action of mutY
• Removes adenine paired to 8-oxoguanine
• Mut name in E. coli comes from mutator
  phenotype (increased spontaneous mutation rate)
  found when any of the gene products is defective

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Post Replication Repair

• Photoreactivation and excision repair are
  short-term responses- occur within minutes of
  damage
• If they are defective or overloaded with
  extensive DNA damage- two other systems act in
  bacteria
• Will be described with regard to thymine dimers
  but also repair other kinds of damage
• Involves recombinational repair and SOS repair
• polymerase cannot replicate past thymine dimer
• It idles at the site
• A gap is left opposite the thymine dimer which
  would be lethal because in the next round of
  replication it would generate a double strand
  break

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Figure 25.15
Recombinational Repair
The undamaged parental strand is transferred to
the gap in the daughter strand, formed by the
inability of DNA polymerase to replicate past it.
Depends on RecA protein important for
recombination and repair it catalyzes strand
pairing. It also acts as a genetic activator,
leading to synthesis of many proteins needed to
adapt to metabolic stress- SOS response The
remaining steps of this process are identical to
those in homologous recombination, which we will
see later.
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SOS response

• Metabolic system that helps the cell survive in
  periods of potentially lethal stresses
• Induced by
• UV irradiation
• Thymine starvation
• Treatment with DNA modifying enzymes
• Inactivation of genes essential to replication
• Responses include
• Mutagenesis
• Since errors in DNA are repaired not by daughter
  strand transfer but by replication- highly error
  prone
• Cell filamentation (elongate but dont divide)
• Activated excision repair
• Why would the cell allow such DNA errors? Better
  to have errors, due to replication past a dimer
  if that is what allows cell to live, than to die

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Mismatch repair

• Mismatches (non-Watson-Crick base pairs) can
  arise through many routes
• Replication error (incorporation of incorrect
  nucleotide)
• Deamination of 5-methylcytosine
• Recombination of DNA segments that are not
  completely homologous
• Sliding of polymerase, creating loops in duplex
  DNA
• Replication error correction
• Error is normally detected by 3 exonucleolytic
  proofreading
• Mismatch repair fixes those that are missed
• mutH, mutL and mutS and DNA helicase in E. coli
• Scans newly replicated DNA looking for mismatches
  and single base insertions or deletions

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Mismatch repair
In eukaryotes there is a similar system,but
recognition step is a bit more complicated -
three different MutS homolog (MSH) proteins form
heterodimers that have different
specialties MSH2-MSH6 recognizes single base
mismatches, insertions and deletion while
MSH2-MSH3 recognizes insertions and deletions of
2-4 nucleotides