Transcript E. coli

DNA repair
Mutación y reparación del DNA
Why repair DNA?
Replication error
• 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
OH.
dUTP
UV hn
H+
mC
TT
P/P
8-oxoG
U
Depurination
All of these events are rare, but the
number of bp in each nucleus is very
large so the total frequency is
significant.
Types of DNA damage
• Base Loss
• Base modification & Deamination
• Chemical Modification
• Photodamage
• Inter-strand crosslinks
• DNA-protein crosslinks
• Strand breakage
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
cell=~3109.
• Rate of breakage of purine glycosidic bonds in
neutral
solution
predicts
=~104
depurinations/day/cell.
• 1013
cells/human
=
1017
depurinations/day/organism!
• 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.
Base loss
Abasic site -loss of a nucleobase
(apurinic or apyrimidinic)
Deamination
Potential Sites of modification/damage
Chemical Damage
Alkylation
Oxidative damage
UV-induced damage
Types of mutations, causes ,
consequences.
• Substitution:
G/C>A/T
(transition);
G/C>C/G
(tranversion). Consequence: usually change the amino
acid sequence if in ORF. Causes: errors in replication;
deamination; oxidation.
• Deletion: GCTAAAAAGCT>GCTAAAAGCT.
• Addition: GCTAAAAAGCT>GCTAAAAAAGCT.
• Consequences: termination of protein synthesis due to
frame-shifting the genetic code. Causes: intercalating
agents; slipped-strand replication errors.
Consequences of mutations (cont)
Accumulation of mutations:
• Cancer, (Loeb’s 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 predisposition to susceptibility to endogenous agents (oxidizing
agents) and environmental insults.
Mutation avoidance
First line of defense:
• Preventing the accumulation of mutationgenerating 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).
Types of Damage Repair
• Photolyase
• De-alkylation proteins (not catalytic)
• Base Excision Repair
• Nucleotide Excision Repair (GG and TC)
• Recombination Repair
• Error-prone Repair
• Double strand Break Repair (if time permits)
Mutation prevention : DNA
repair
Evidence for repair
mechanisms:
Evelyn Witkin: UV
light,
death,
mutagenesis, and
survival.
1.0
0.1
S/So 0.01
0.001
Mutation
frequency
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?
Interpretation of the UV survival
~100
curve
than one
More
“hit” is required
to
kill
an
organism
UV damage is repaired efficiently but
some damage is mutagenic
1.0
0.1
Repair can’t keep up with
damage; mutants too are killed
0.01
S/So
0.001
Mutation
frequency
0.0001
S=survival;
So=survival before
UV treatment
UV dose
polA (DNA polI mutants)
Rare
mutants
are UVresistant.
Could low does protect against
cancer-the hormesis hypothesis: data
for and against
100
Linear response
cancers
extrapolated to zero dose-is this
an appropriate extrapolation?
0
0
10
dose
100
1000
Radiation-sensitive mutants
are easy to identify
• E. coli~ 30
genes
are
involved in
DNA repair.
Yeast~
50
genes.
• Humans?
-UV
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
One colony is missing
because the cells are
more UV-sensitive than
those in other colonies
Redundancy of repair
mechanisms
•
•
•
•
•
•
•
1. Proof-reading or editing by DNA polymerase
2. Direct reversal of damage.
3. Base excision repair
4. Nucleotide excision repair*
5. Mismatch repair.
6. Error-prone (SOS) repair*
7. Recombination repair
* Induced by DNA damaging agents
Editing: Frequency of errors in
replication depends on the polymerase
Orga nism
Polymerase
Error rate
3Υexo?
(chang es/base/gene ration)
RNA vir us (HIV)
10-4
-
T4 DNA Pol
10-7
+
DNA
10-8
+
Rever se
transc riptase
DNA vir use s
E.
coli,
yea st,
Drosophil a, humans
Mutation rate in vivo
PolIII-
li ke
10-10
DNA repair polymerases
DNA polymerase Eta (XP-V) - addition of two dA residues
across pyrimidine dimers
DNA polymerase Zeta - addition of random residues
across pyrimidine dimers
Direct removal of 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.
Enzymatic reversal of T-T dimer
formation
DNA photolyase is present in
all organisms. Which cells
in your body do you think
would have the most
photolyase?
300-500 nm light
AGCATTCTGA
TCGTAAGACT
200-300 nm light
AGCATTCTGA
TCGTAAGACT
AGCA{T/T}CTGA
TCGT {AA} GACT
DNA photolyase
Direct reversal of DNA damage:
No excision of bases or nucleotides
Alkyltransferase detoxification of
alkylated DNA by the Ada protein
• A second type of direct reversal of
DNA damage removes offending
akyl groups from O6-alkyl guanine
and methylated phosphate triesters.
Alkylation of Cys321 inactivates
the protein-the protein “commits
suicide”.
Buried active site cysteine321
covalently binds methyl group
of O6mG.
Base Excision Repair
Base Excision Repair
Nucleotide Excision Repair
(E.coli)
Nucleotide Excision Repair
(Global Genome Repair -Humans)
Nucleotide Excision Repair
(Transcription Coupled -Humans)
Common features of GGR & TCR
4. DNA glycosylases remove altered bases
Deamination
of
cytosine,
particularly 5-methyl cytosine
leaves uracil (thymine if 5methyl cytosine) in the DNA.
Uracil would pair as thymine
during replication and thus
cause a mutation.
Uracil-N-glycosylase removes
uracil from DNA.
An endonuclease then cleaves
the backbone at that site,
creating a substrate for NER
5. DNA methylation and repair
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
The DNA methyl transferases lag
several thousand bases behind
the replication fork. This “marks”
the parental DNA strand.
= methyl group
N6-methyl-adenine
Mismatch repair corrects
errors occurring during
DNA replication
Mismatch
correction
accounts
for
the
“discrepancy” between the
error rate of polIII in vitro
and error rates measured in
vivo.
Mismatch repair
corrects the
unmethylated
strand
What happens if the “old” strand needs
repair? eg 5-methyl cytosine>deamination 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.
Mis-paired bases
Mismatch repair-the finale
6. Error-prone or induced DNA repair- the
SOS reponse
UV’d T4 phage
UV
UV’d T4 phage
E. coli
E. coli
Higher frequency of surviving
phage, but many mutants.
• 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
Few surviving phage
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
A’s.
AGCTAGTCAT/TCAGTC
Replication stops
at T/T dimer
SOS response:
AGCTAGTCAT /TCAGTC
TCGATCANNNNGTCAG
Error-prone polymerase allows
replication to proceed, albeit
inaccurately
Error Prone Bypass
(E. coli)
Experimental evidence for Error prone repair
(E.coli)
Revertant in His- genes
(umuC mutated strain)
UV-responsive activation of
the umuC gene
Human mismatch repair genes and
cancer
• Yeast mutants defective in mismatch
repair
genes
have
unstable
microsatellite sequences (repetitive
tracts of mono- and dinucleotides).
• Some human colon cancers also
display microsatellite instability.
• Are these due to defective mismatch
repair genes?
ANSWER: Yes: Genetic
mapping of human nonpolyposis colon cancer
genes identifies these
genes as defective
human mismatch repair
genes.
Can these be corrected?
…..
Model for activation of DNA damage repair
Damage & Repair
• Multiple forms of DNA damage occur
• These are repaired constantly by several
mechanisms
• Failure to repair damage leads to mutations
• Often defects in damage sensing machinery
or DNA repair processes can be correlated
with increased incidence of diseases such as
cancer
Factors involved in Damage Sensing
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.