2. Metabolic Responses to DNA damage

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2. Metabolic Responses to DNA damage

• DNA damage
• I. Deamination of 5-methyl cytosine and
  cytosine can produce mutations
• II. UV damage of DNA
• B. Types of DNA repair
• I. Direct repair
• II. Nucleotide excision repair
• III. Base excision repair
• IV. Postreplication repair
• V. Mismatch repair

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A. DNA Damage I. Deamination of methylated
cytosine can result in base pair changes
- mismatches can arise from the deamination of
5-methyl-cytosine (and 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
Corrected by mismatch repair pathway (see later)
N
O
N
O
H2O - NH3
5-methyl cytosine
thymine
Oxidative deamination e.g. by nitrous acid
H
H
O
N
H
Corrected by uracil-DNA N-glycosylase pathway
N
U
N
C
N
O
N
O
uracil
cytosine
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A. DNA DamageII. UV Damage of DNA

• A common type of DNA damage is associated with
  exposure
• to Ultraviolet Light
• - can produce 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 either cyclobutane rings involving carbons 5
  and 6 or 6-4 carbon linkages
• Ability of an organism to survive UV irradiation
  directly correlates with its ability to remove
  thymine dimers from its 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
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B. Types of Repair

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

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I. Direct repair

• b. Repair of alkylation damage by O6-alkylguanine
  alkyltransferase
• Alkylation of DNA can block DNA replication
  because of modified bases that are formed
• Caused by alkylating agents-sometime used in
  cancer 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-methyl guanine
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Mispairing of O6-methylguanine with
thymine Creates 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 proteins active site The
protein can only function once-it gets turned
over by the cell- so its not really an enzyme
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B. Types of Repair II. Nucleotide Excision
Repair
Excision repair of thymine dimers by E. coli
UvrABC excinuclease - 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,
DNA polymerase I and ligase remove the damaged
DNA and replace it with new DNA Enzyme not a
typical endonuclease because it cuts at 2
distinct sites- therefore termed an excinuclease
for its role in excision repair
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III. Base excision repair (BER)

• Also removes one or more nucleotides from site of
  base damage
• Initiates with cleavage of glycosidic bond
  between damaged base and deoxyribose
• Also functions in removal of thymine dimers and
  uracil (for a description of the U-specific
  process, see lecture 12)
• e.g. Endonuclease V of Bacteriophage T4
• Has two activities
• A. Glycosylase- cleaves between thymine on 5
  side of dimer and its deoxyribose
• B. AP endonuclease- recognizes apyrimidinic (AP)
  site- consists of deoxy ribose without an
  associated pyrimidine base, cleaves on the 5
  side
• C. Next, deoxyribo-phosphodiesterase in cell
  cleaves 3 to AP site
• D. 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 (see Fig. 25.13 of Mathews)

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Oxidative damage of DNA is usually repaired by BER

• Example of oxidative damage conversion of
  adenine to 8-oxoguanine
• Highly mutagenic because it can base pair with
  adenine
• In E. coli, repair can occur through action of
  three gene products
• 1. mutT product cleaves base before DNA
  incorporation
• 2. Actual repair can occur through action of
  mutM product removes 8-oxoguanine from DNA
• 3. Or, repair can occur through action of
  mutY product
• removes adenine paired to 8-oxoguanine-allows
  chance of repair in next round of replication.
• 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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IV. Post Replication Repair

• Direct repair and excision repair systems involve
  rather short-term responses- occur within minutes
  of damage
• If they are defective or overloaded with
  extensive DNA damage- two other longer term
  systems act in bacteria
• Will be described with regard to thymine dimers
  but also repair other kinds of damage
• During replication, DNA polymerase III cannot
  continue to replicate a strand containing a
  thymine dimer and so
• 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
• There are two mechanisms for gap repair 1)
  recombi-national repair and 2) SOS repair or
  error-prone repair.

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Figure 25.15
Recombinational Repair
The undamaged parental strand is transferred to
the gap (opposite the damaged site) in the other
duplex, caused by the inability of DNA
polymerase to replicate past the dimer. Depends
on RecA protein important for recombination and
repair it catalyzes strand pairing or
heteroduplex formation 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 alarm system that helps the cell
  survive in periods of potentially lethal stresses
• Induced by
• UV irradiation
• Thymine starvation
• Treatment with DNA modifying agents
• Inactivation of genes essential to replication
• Responses include
• Mutagenesis associated with error-prone repair
• -Gaps opposite DNA damage are filled by
  replication- but it is highly error prone DNA
  pol III is aided in replication by action of
  several proteins, including RecA
• Cell filamentation (elongate but dont divide)
• Activation of 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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V. Mismatch repair

• Mismatches (non-Watson-Crick base pairs) can
  arise through many routes
• - Replication errors (incorporation of incorrect
  nucleotides or tautomeric change)
• Deamination of 5-methylcytosine
• Recombination of DNA segments that are not
  completely homologous
• - Sliding of polymerase, creating loops or
  bulges in duplex DNA
• Correction of replication error/deamination of
  5-mC is best understood
• - Replication errors normally
  detected by 3 to 5 exonucleolytic
  
• proofreading of DNA
  polymerases
• - Mismatch repair fixes rep.
  errors that are missed and problems
• associated with deamination
  of 5-mC.

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Mismatch Repair via proofreading
DNA polymerases I, II, III have 3 ? 5
exonuclease editing function DNA polymerase I
also has 5 ? 3 exonuclease activity HOW do
most of these replication errors occur in the
first place? Natural rate of misincorporation
1 in 10,000 bases This usually arises due to
base Tautomerism
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Tautomerism

• the existence of a particular compound in two or
  more distinct forms

KETO - Guanine
ENOL - Guanine
( normal )
10,000 1 at equilibrium (i.e. frequency 10-4)
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Tautomers
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Mismatches overlooked by proofreading and 5-mC
defects are dealt with by mismatch repair system

• mutH, mutL and mutS and DNA helicase in E. coli
• Scans newly replicated DNA looking for
  mismatches and single base insertions or
  deletions cuts out part of one strand containing
  mismatch and this is replaced by DNA polymerase
  III and linked to rest of strand via DNA ligase

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If the resulting mismatch is not immediately
repaired during synthesis, how does the cell know
which strand to repair? The cell
machinery can determine which is the newly
synthesized strand and which is the template
strand The template strand is methylated, which
makes it possible to identify it
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Eukaryotic 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