Lectures 78:

1
Lectures 7-8 DNA stability Damage and
repair M. Schultze
Learning outcomes Mechanisms by which DNA
damage may lead to mutations Mechanisms by which
damaged DNA can be repaired
2
The good and the bad of mutations
Horticultural varieties of Potentila fructicosa
Skin cancer in Xeroderma pigmentosum
3
Mutations Changes in DNA sequence

• Point mutations
• Single base changes, 1-2 bp insertions or
  deletions

Silent/Synonymous GGC (Gly) ? GGT
(Gly) Missense (conservative) GAC (Asp)? GAA
(Glu) Missense (nonconservative) GGC (Gly) ?
GTC (Val) Nonsense AGA (Arg) ? TGA
(stop) Frameshift ACC GGC AGA ? ACC GGA GA

• Large ( 3 bp) deletions or insertions

4
Spontaneous mutations
5
Replication errors Wrong bases put to the
wrong place
6
Mispairing of tautomers
AImino
TKeto
CImino
TEnol
GEnol
7
Transient occurrence of bases in their enol form
Results in GC ? AT or AT ? GC
A T
Tenol
A
A T
G
G
Tenol
Tketo
Repair
Transition purine replaced by purine,
pyrimidine by pyrimidine
A T
G C
8
Small deletions or insertions caused by base
slipping
Click here to see animation, or go to web site of
Griffiths, animation 14-21 (15-10 in 9th ed)
(From Griffiths, Introduction to Genetic
Analysis, 8th ed.) http//bcs.whfreeman.com/iga8e/
9
Example of frameshift mutation
The reading frame behind the insertion site is
changed!
10
Spontaneous lesions
11
Loss of bases
Apurinic or Apyrimidinic sites AP sites In
humans at body temperature 10,000 bases lost per
cell per day!
12
AP sites may cause mutations if not repaired
G C
C
Repair
Missing information on template DNA. Random
incorporation of bases, frequently A!
G C
A
T A
13
Spontaneous deamination
Uracil
Cytosine
5-methyl cytosine
Thymine
14
5-methyl cytosine mutational hot spots in the
lacI gene of E. coli
Red 5-methyl-C White normal C
(From Griffiths, Introduction to Genetic
Analysis, 8th ed.)
15
Base pairing between 8-oxo-guanine and adenine
A
Rib
Rib
8-oxo-G
16
How 8-oxo-G can cause mutations
ROS
C
8-oxo-G
C
G
Results in GC ? TA or AT ? CG
8-oxo-G A
G
Repair
C
Transversion Purine replaced by
pyrimidine, pyrimidine by purine
Repair
T A
8-oxo-G A
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Induced mutations
18
Alteration of bases for example - Deamination
by nitrous acid - Alkylating agents (e.g. EMS)
- Aflatoxin, benzo(a)pyrene
19
Mutation by alkylating agents
G
Ethylmethane sulfonate (EMS)
T
(Results in GC ? AT transitions)
O-6-Ethylguanine
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Base damage through bulky adducts
Aflatoxin B1
Benzo(a)pyrene
21
How aflatoxin can cause mutations
aflatoxin
C
G
C
G
Replication arrest upon addition of C
G
A
C
Replication bypass upon addition of A
DNA polymerase
GC ? TA transversion
T A
G C
next round of replication
22
Metabolic conversion of benzo(a)pyrene into a
mutagen
Diol epoxide
23
Ames test
Paper disc
Compound X Liver extract
Minimal medium Salmonella His-
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Intercalating agents stain nucleic acids
Acridine orange
Proflavin
Ethidium
(Results in deletions and insertions)
25
UV induces pyrimidine dimers
Replication arrest bypassed (SOS in bacteria)
UV
CG ? TA transition
e.g. strong sun light on beach 40,000 dimers per
epidermal cell per hour!
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Section Summary

• Spontaneous mutations
• Replication errors (base changes, deletions,
  insertions)
• Spontaneous lesions (Loss of bases, deamination,
  oxidation)
• Induced mutations
• Alteration of bases (base changes)
• Intercalating agents (deletions/insertions)
• UV radiation (pyrimidine dimers)
• Ionising radiation (strand breakage, loss of
  bases, oxidation)

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DNA repair
28

• Prevention of DNA damage
• Proofreading by DNA polymerase
• Hydrolysis of damaged nucleotides
• (e.g. 8-oxoGTP ? 8-oxoGMP)

29

• Direct reversal of DNA damage
• Photoreactivation (reversal of pyrimidine
  dimers)
• Alkyltransferases
• (e.g. O6-methylguanine-DNA methyltransferase,
  MGMT,
• removes methyl group)

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Excision repair Three mechanisms
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Base excision repair (BER)
Uracil DNA glycosylase
Many other DNA glycosylases, e.g. 8-oxoguanine
DNA glycosylase (OGG1)
AP site
AP endonuclease
AP lyase (removes deoxyribosephosphate)
DNA polymerase (fills gap with correct
nucleotide)
DNA ligase (joins the ends)
32
Nucleotide excision repair (NER)
BP
Recognition of base damage by repair complex
G
C
BP
G
Incision of DNA strand on both sides
C
C
Removal of oligonucleotide
Filling of gap by DNA polymerase
C
G
Joining of ends by DNA ligase
C
33
MutS
MutL
Mismatch repair (MMR)
MutS
MutH
Exonuclease
DNA polymerase
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Repair of double-strand breaks
35
Nonhomologous end-joining (NHEJ)
Binding of ends by protein complex
Trimming of ends
(From Griffiths, Introduction to Genetic
Analysis, 8th ed.)
Joining of ends
Results in the loss of some bases!
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Accurate repair of double-strand breaks by
homologous recombination (HR)
One strand of broken DNA invades intact DNA strand
Sequence information recovered across the gap
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p53 plays a key role in co-ordinating replication
and repair
- guardian of the cell - Defective p53
associated with 50 of human cancers
Cell cycle arrest until damage is repaired
Too late for arrest, Apoptosis induced cell
death
38
Section Summary
Measures against DNA damage

• Prevention of damage
• Direct reversal
• Excision repair systems
• Double-strand break repair

Repair and replication must be co-ordinated