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DNA Damage, Mutations, and Repair See Stryer p. 768-773
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DNA Damage, Mutations, and Repair See Stryer p. 768-773.

Dec 22, 2015

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Page 1: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

DNA Damage, Mutations, and Repair

See Stryer p. 768-773

Page 2: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 3: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 4: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 5: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 6: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 7: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 8: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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)

Page 9: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 10: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 11: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

Deletions and insertions can be caused by intercalating agents

Stryer Fig. 27.44

Page 12: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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.

Page 13: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

DNA Repair Types

• Direct repair– Alkylguanine transferase– Photolyase

• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair

• Recombination repair

Page 14: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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)

Page 15: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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.

Page 16: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 17: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 18: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 19: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 20: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 21: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

DNA Repair Types

• Direct repair– Alkylguanine transferase– Photolyase

• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair

• Recombination repair

Page 22: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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)

Page 23: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 24: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 25: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 26: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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.

Page 27: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 28: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

• 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

Page 29: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 30: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 31: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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

Page 32: DNA Damage, Mutations, and Repair See Stryer p. 768-773.

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