DNA Damage, Mutations, and Repair See Stryer p. 768-773.
Post on 22-Dec-2015
217 Views
Preview:
Transcript
DNA Mutations
1. Substitution mutations: one base pair for another, e.g. T for G• the most common form of mutation
• transitions; purine to purine and pyrimidine to pyrimidine
• transversions; purine to pyrimidine or pyrimidine to purine
2. Frameshift mutations
• Deletion of one or more base pairs
• Insertion of one or more base pairs
Rare imino tautomer of A
N N
NH2
O
HN
NN
N
NH
C
• Very low rate of misincorporation (1 per 108 - 1 per 1010)• Errors can occur due to the presence of minor tautomers
of nucleobases.
Spontaneous mutations due to DNA polymerase errors
N
N
N
N
H2N
NH
N
O
O
AT
H3C
Normal base pairing Mispairing
10-4
amino
A(imino)T
AT
A(imino)C
AT
GC
Final result: A G transition (same as T C in the other strand)
Consider misincorporation due to a rare tautomer of A
AT
1st
replication
5’3’
Normal replication
2nd replication
Induced mutations result from DNA damage
Sources of DNA damage: endogenous
1. Deamination2. Depurination: 2,000 - 10,000 lesions/cell/day3. Oxidative stress: 10,000 lesions/cell/day
Sources of DNA damage: environmental
1. Alkylating agents2. X-ray 3. Dietary carcinogens4. UV light 5. Smoking
N
NH
NN
O
NH2
N
N
NH2
O
G C
o
h
h
HN
NH
O
ON
N
NN
OR
NH2
TO6-AlkG
n
h
G A
GC
GT
AT
Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase
DNA oxidation
H3CNH
N
O
O
H3CNH
N
O
O
HO
HO
thymine glycol
NNH
NN
O
NH2
HN
NH
NN
O
NH2
O
8-oxo-G
Reactive oxygen species: HO•, H2O2, 1O2, LOO•
•10,000 oxidative lesions/cell/day in humans
N N
NN
NH2
NNH
NN
O
NH2
N
N
NH2
O
N NH
NN
O
NNH
NH
N
O
O
NH
N
O
O
Hypoxanthine
Xanthine
Uracil
NNH
NN
O
N
N
NH
O
H
A G
Deamination
N N
NN
NH2
HO
N NH
NN
NH2HO
N NH
NN
O
Mechanism:
H2O
- NH3
A
G
C
C
Rates increased by the presence of NO (nitric oxide)
Depurination to abasic sites
N NH
NN
O
NH2O
O
O
OHOO
O
Abasic site (AP site)
H2O
N NH
NNH
O
NH2
2,000 – 10,000/cell/day
UV light-induced DNA Damage
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
…CC… Pyrimidine dimer
Easily bypassed by Pol (eta) in an error-free manner
Importance of DNA Repair
• DNA is the only biological macromolecule
that is repaired. All others are replaced.
• More than 100 genes are required for DNA repair, even in organisms with very small genomes.
• Cancer is a consequence of inadequate DNA repair.
DNA Repair Types
• Direct repair– Alkylguanine transferase– Photolyase
• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair
• Recombination repair
N N
NN
O
NH2
CH3
O6-methylguanine
AGT-CH2-SH
N NH
NN
O
NH2
AGT-CH2-S CH3
Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.
Direct repair: O6-alkylguanine DNA alkyltransferase (AGT)
Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same genetic information
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
A ::
G
T ::
T
A
3'
3' 5'
5'
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
Original DNA duplex DNA duplex with one of the nucleotidesremoved
Repaired DNA duplex
Base excision repair (BER)
• Used for repair of small damaged bases in DNA (AP sites, methylated bases, oxidized bases…)
• Human BER gene hogg1 is frequently deleted in lung cancer
HN
NH
NN
O
NH2
O
8-oxo-G
OHOO
O
Abasic site (AP site)
NNH
NH
N
O
O
XanthineN N
NN
NH2
Me
N3-Me-Ade
Nucleotide Excision Repair
• Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
5'
3'
NH
NH
NN
NO
HO
HOOH
HOO
OH
• Xeroderma pigmentosum is a genetic disorder resulting in defective NER
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base)
Parental strand is methylated:
2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)
N
N
NH2
O
H3CN N
NN
HNMe
Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum NER
Hereditary nonpolyposis MMRcolorectal cancer
Cockrayne’s syndrome NER
Falconi’s anemia DNA ligase
Bloom’s syndrome BER, ligase
Lung cancer (?) BER
DNA Repair Types
• Direct repair– Alkylguanine transferase– Photolyase
• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair
• Recombination repair
N N
NN
O
NH2
CH3
O6-methylguanine
AGT-CH2-SH
N NH
NN
O
NH2
AGT-CH2-S CH3
Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.
Direct repair: O6-alkylguanine DNA alkyltransferase (AGT)
AGT inhibitor O6-benzylguanine is in clinical trials to be used in conjunction with antitumor alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
tumor cell death
AGT overexpression in tumors makes them resistant to alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
AGT
normal dG
tumor cells survive
Combination therapy with O6-benzylguanine overcomes tumor resistance to alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
tumor cell deathAGT
normal dG
N
N
N
O
NH2NH
Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same genetic information
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
A ::
G
T ::
T
A
3'
3' 5'
5'
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
Original DNA duplex DNA duplex with one of the nucleotidesremoved
Repaired DNA duplex
• Used for repair of small damaged bases in DNA (AP sites, methylated bases, deaminated bases, oxidized bases…)
• Human BER gene hogg1 is frequently deleted in lung cancer
Base excision repair (BER)
HN
NH
NN
O
NH2
O
8-oxo-G
OHOO
O
Abasic site (AP site)
NNH
NH
N
O
O
Xanthine
N N
NN
NH2
Me
N3-Me-Ade
Uracil DNA glycosylase removes deaminated C
N
N
NH2
O
NH
N
O
O
Uracil
BERC
Not normally present in DNA
No Me group
Cytosine
Thymine
H3CNH
N
O
O
Normal DNA baseNot recognized by BER
Nucleotide Excision Repair
• Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
5'
3'
NH
NH
NN
NO
HO
HOOH
HOO
OH
• Xeroderma pigmentosum is a genetic disorder resulting in defective NER
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base)
Parental strand is methylated:
2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)
N
N
NH2
O
H3CN N
NN
HNMe
G T
top related