Biochemistry 441 Lecture 13 Ted Young March 8, 2000

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Biochemistry 441 Lecture 9 Ted
YoungJanuary 28, 2008

• Topic for today
• DNA repair

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• Experience, the universal Mother of Sciences
• Miguel Cervantes, Don Quixote

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Why repair DNA?

• 1. Errors in DNA replication
• 2. Endogenous DNA damage and mutagens
• 3. Environmental insults to DNA
• 4. Un-repaired damage leads to
• -mistakes in RNA/protein synthesis
• -inherited as genetic alteration-a mutation
• -death

Replication error
OH.
H
dUTP
UV
hn
mC
8-oxoG
TT
Depur- ination
P/P
U
All of these events are rare, but the number of
bp in each nucleus is very large so the total
frequency is significant.
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Example of the product of a very small and a very
large number yielding a significant effect

• Number of bp in the nucleus of a human
  cell6X10e9.
• Example 1 Rate of breakage of purine glycosidic
  bonds in neutral solution predicts 10e4
  depurinations/day/cell. X10e13 cells/human
  10e17 depurinations/day/organism! Example 2 UV
  in sunlight causes 50 thymine dimers/sec/cell .
• If these are not repaired, it would lead to
  massive errors in the synthesis of proteins.
  Mutations in the germ line would transmitted to
  offspring, leading to genetic disease.

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Consequences of mutations (cont)

• Accumulation of mutations
• -cancer (Loebs hypothesis) cancer is a genetic
  disease caused by an elevated mutation rate-as
  by an error-prone polymerase or faulty repair
  machinery. Two-hit model. Somatic versus germ
  line mutations.
• -aging (error catastrophe hypothesis) failure
  of normal cell death (apoptosis) due to
  accumulation of mutations in genes responsible
  for the normal operation of these processes.
• Single mutations and genetic disease-how many
  genes, when inactivated, would cause a disease?
  
• Multiple polymorphisms (remember SNPs) and
  pre-disposition to susceptibility to endogenous
  agents (oxidizing agents) and environmental
  insults.

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Mutation avoidance

• First line of defense
• Preventing the accumulation of mutation-
  generating agents metabolism of active oxygen
  species by reducing agents and enzymatic
  mechanisms (superoxide dismutase) dUTPase to
  prevent mis-incorporation of dUMP into DNA
  shielding from harmful irradiation (melanin in
  skin).

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Mutation prevention DNA repair

• Evidence for repair mechanisms
• Evelyn Witkin UV light, death, mutagenesis, and
  survival.

1.0
0.1
S/So
0.01
Mutation frequency
0.001
0.0001
UV dose
Note the dose-response curve is not linear at
low doses there is high survival at higher doses
survival drops off rapidly at very high doses
there is more resistance. Mutations occur at
increasing frequency, and then decline. Why the
decline?
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Interpretation of the UV survival curve
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• More than one hit is required to kill an
  organism

UV damage is repaired efficiently but some damage
is mutagenic
Repair cant keep up with damage mutants too are
killed
1.0
0.1
S/So
0.01
Mutation frequency
0.001
0.0001
Rare mutants are UV- resistant.
Ssurvival Sosurvival before UV treatment
UV dose
polA (DNA polI mutants)
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Could low dose protect against cancer? The
hormesis hypothesis
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Linear response
cancers
extrapolated to zero/(low) dose-is this an
appropriate extrapolation?
0
0 50 100 150
dose
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Radiation-sensitive mutants are easy to identify

• E. coli 30 genes are involved in DNA repair.
  Yeast 50 genes.
• Humans?

UV light-individual cells on a petri plate
to induce mutations
Grow to colonies
Replica plate using a piece of velvet to pick-up
and transfer colonies to a new petri plate.
UV
-UV
One colony is missing because the cells are more
UV-sensitive than those in other colonies
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Redundancy of repair mechanisms

• 1. Proof-reading or editing by DNA polymerase
• 2. Mismatch repair.
• 3. Base excision repair
• 4. Nucleotide excision repair
• 5. Direct reversal of damage.
• 6. Error-prone (SOS) repair
• 7. Recombination repair
• Induced by DNA damaging agents

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1. Editing Frequency of errors in replication
depends on the polymerase

• Organism Polymerase Error rate
  (changes/ base/generation)
• RNA virus (HIV) Reverse transcriptase 10-4 -
• DNA viruses T4 DNA Pol 10-7
• E. coli, yeast, DNA PolIII-like 10-8
  Drosophila, humans
• Mutation rate in vivo 10-10

3 exo?
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2. DNA methylation and repair
The DNA methyl transferases lag several thousand
bases behind the replication fork. This marks
the parental DNA strand.

• Methylation occurs on cytosine and adenine
  residues in DNA. N6 of A is methylated in the
  sequence GmATC C is methylated in the sequence
  CmC(A/T)GG

methyl group
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Mismatch repair corrects errors occurring during
DNA replication
N6-methyl-adenine

• Mismatch correction accounts for the
  discrepancy between the error rate of polIII in
  vitro and error rates measured in vivo.

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Mismatch repair corrects the unmethylated strand
Mis-paired bases
What happens if the old strand needs repair? eg
5-methyl cytosinegtdeamination to 5-methyl uracil
(thymine!). In E. coli a small fraction of C is
5-methylated and these are hot-spots
for spontaneous mutation. This implies that
5-methyl cytosine is frequently either not
repaired or is mistakenly repaired on the wrong
strand.
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Mismatch repair-the finale
Homologous enzymes to MutS, L, and H exist in
eukaryotes but the signal for distinguishing old
versus new strands is different.
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3. DNA glycosylases remove altered bases

• Deamination of cytosine, particularly 5-methyl
  cytosine leaves uracil (thymine if 5-methyl
  cytosine is present) in the DNA. Uracil would
  pair as thymine during replication and thus cause
  a mutation. The repair enzyme uracil-N-glycosylase
  removes uracil from DNA. An endonuclease then
  cleaves the backbone at that site, creating a
  substrate for DNA pol I and subsequent
  restoration of strand integrity by DNA ligase.

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4. Nucleotide excision repair (NER)

• Another major type of repair is ubiquitous-being
  found in all organisms. Several rare human
  disorders are caused by defects in NER. Most of
  the genes identified are involved directly in
  repair. Some, however, participate in
  transcription instead, coupling the two processes
  mechanistically.

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Ultraviolet light and DNA damage T-T dimers

• A common photoproduct of UV treatment of DNA in
  vivo and in vitro is an intra-strand dimer formed
  beween adjacent thymines.

Note that formation of the cyclobutane
ring destroys the aromatic nature of the
pyrimidine ring and distorts the helix.
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5. Direct removal of damage

• Removal of thymine-thymine dimers by a photolyase
  enzyme that is light-activated
• A second type of direct reversal of DNA damage
  removes potentially mutagenic alkyl groups from
  O6-alkyl guanine and methylated phosphate
  triesters. Alkylation of Cys321 inactivates the
  protein-the protein commits suicide.

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6. Error-prone repair-the SOS reponse
UVd T4 phage
UVd T4 phage
UV
E. coli
E. coli
Higher frequency of surviving phage, but many
mutants.
Few surviving phage

• Irradiation of bacteria before virus infection
  enhanced repair of damaged viral genes but led to
  mutations. This has an evolutionary advantage for
  the viral population since it increases the
  probability that some members will survive albeit
  in altered form

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Error-prone repair is due to a novel
damage-induced DNA polymerase activity

• Two genes induced by cleavage of LexA (a DNA
  binding repressor protein), umuC and umuC, encode
  a DNA polymerase activity that is active on
  damaged DNA templates-ie templates lacking a
  proper DNA sequence. It allows replication past
  the damaged site, often inserting (incorrectly)
  one or a few As.

AGCTAGTCAT/TCAGTC
Replication stops at T/T dimer
SOS response
AGCTAGTCAT/TCAGTC TCGATCANNNNGTCAG
Error-prone polymerase allows replication to
proceed, albeit inaccurately
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Recombination can also repair DNA damage
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Human repair genes and cancer

• Defects in mismatch repair, NER, error-prone
  repair, and trans-lesion bypass have all been
  associated with (meaning they cause) human
  cancers.
• Read Box 25-1 in your text for more information.

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DNA Repair-summary

• DNA repair mechanisms exist in all organisms to
  maintain the fidelity of the DNA sequence.
• DNA is repaired during and after replication, and
  by constitutive and damage-inducible enzyme
  systems
• Multiple repair mechanisms are necessary to
  correct errors arising during replication and to
  repair DNA damage by intrinsic and extrinsic
  agents.
• Failure of DNA repair leads to mutations and
  cancer. The disease (or disease
  predisposition)may be hereditary if the mutation
  occurs in a germ cell or germ cell precursor or
  it may occur in a somatic cell, leading to a
  non-hereditary form of disease-cancer or
  otherwise.