The Virtual Free Radical School
DNA Oxidation: A Simple
Overview
Freya Q. Schafer, Ph.D.
Free Radical and Radiation Biology Program
Department of Radiation Oncology
The University of Iowa
Iowa City, IA 52242-1101
Tel: 319-335-6749
Email: freya-schafer@uiowa.edu
DNA Oxidation
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Schafer 1
Components of
DNA and RNA
Pyr im idin es:
O
NH 2
CH 3
HN
O
N
N
H
O
t h ym in e
CH 3
N
N
H
O
cyt osin e
O
NH 2
HN
O
N
H
5-m et h ylcyt osin e
H O CH 2
H
N
H
H
OH
-D-2-Deoxyr ibose
H O CH 2
OH
O
O
H
N
N
HN
H
N
OH
H
H
u r acil
Pu r in es
NH 2
OH
O
N
N
H
aden in e
H
H2N
N
N
H
gu an in e
H
OH
-D-Ribose
sugars
bases
DNA Oxidation
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phosphate back bone
DNA-Structure adapted from:
National Human Genome Research
Institute (NHGRI); by artist Darryl Leja
http://www.nhgri.nih.gov/DIR/VIP/
Schafer 2
Hot Spots for Free Radical Attack
O
-
N7
O
O P O CH 2
-O
4'
3'
4
8 9
3
NH 2
O
1'
2'
HN3
O
2
O P O CH 2 O
O
O
4'
DNA Oxidation
1 NH
2
N
N
O
6
5
3'OH
CH 3
4
5
6
1
N
1'
2'
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Schafer 3
Oxidative Damage to the Bases
O
HO• attack on pyrimidines
HN 3 4 5
OH
CH 3
.H
reducing
21 6
O
O
O
CH 3
HN
+
O
N
H
N
H
.
HO
H
HN
O
N
H
O
HN
O
DNA Oxidation
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CH 3
oxidizing
Thymine
Adapted from: von Sonntag C. (1987)
The Chemical Basis of Radiation Biology.
Taylor & Francis London, NY.
.
N
H
H
OH
.CH
2
H
Schafer 4
Oxidative Damage to the Bases
O
HO• attack on purines
2
H 2N
O
N
+ HO
N
3
N
HO
N
H
H
N
H
.
H
OH
HN
H 2N
N
.N
N
H
H
O
Guanine
HN
Adapted from: von Sonntag C. (1987)
The Chemical Basis of Radiation Biology.
Taylor & Francis London, NY.
DNA Oxidation
4
7N
9 8
O
HN
H 2N
.
HN1 6 5
H 2N
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N
.N
H
N
OH
H
Schafer 5
NH2
N
H
N
N
N
H
HN
N
OH
N
H2N
N
O
O
N
H
OH
O
N
H
N
H
O
5-hydroxymethyluracil
OH
N
H
H
O
H
5-hydroxy-6-hydrouracil
5-hydroxyuracil
H2N
NH2
N
4,6-Diamino-5formamidopyrimidine
NH CHO
O
2,6-Diamino-4-hydroxyformamidopyrimidine
N
N
N
H
CH3
OH
OH
H
HN
N
H
Thymine glycol
(cis and trans)
NH2
NH
O
CH3
H
H
H
N
H
5,6-dihydrothymine
O
N
N
NH2
H
OH
8,5'-cyclo-2'-deoxyadenosine
NH2
H
H
H
DNA Oxidation
O
N
H
5-hydroxy-6hydrothymine
HO
H
H
HN
O
O
HO
N
CH3
OH
H
H
HN
NH2
N
NH2
N
O
O
HN
NH CHO
OH
N
H
O
N
O
OH
HN
NH2
H
Adapted from: Dizdaroglu M. (1992)
Free Radic Biol Med. 10:225-242.
Dizdaroglu M. (2002)
Free Radic Biol Med. 32:1102-1115.
O
H
HN
H
OH
5-hydroxy
hydantoin
O
CH2OH
O
HN
8-hydroxyguanine
8-hydroxyadenine
HN
Examples of Oxidized
DNA Bases
O
H
8,5'-cyclo-2'-deoxyguanosine
N
O
OH
OH
N
H
H
Cytosine glycol
N
O
O
OH
N
H
5-hydroxycytosine
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HN
OH
N
H
5,6-dihydroxy
uracil
O
HN
OH
O
CH3
N
OH
H
5-hydroxy-5methylhydantoin
O
Schafer 6
Oxidation of DNA can lead to Mutations
8-h ydr oxygu an in e
O
O
HN
N
N
Basepair s
HN
N
NH
A T
H2N
N
N
H
OH
H2N
8 OH-Gu a (en ol)
H
N
H2N
O
N
H
H N
O
H
NH
N
N
H
N
N
H
DNA Oxidation
N
H
8 oxo-Gu a (k et o)
N
8 OH-Gu a
N
H
G C
8 OHdG A
Misreading of 8OHdGua can lead
mutation (GCAT
transversion).
Aden in e
O
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Schafer 7
Oxidation of 5-methylcytosine leads to
Rapid Deamination.
NH 2
CH 3
N
O
NH 2
CH 3
OH
H
N
oxidat ion
N
NH 2
O
N
DNA
en don u clease III
O
OH
DNA
CH 3
OH
H
N
N
H
5-m et h ylcyt osin e glycol
5-m et h ylcyt osin e
H2O
H2O
NH 3
NH 3
O
H
O
OH
N
N
DNA
O
CH 3
OH en don u clease III
H
H
OH
O
CH 3
OH
H
N
N
Deamination
can result in:
a) loss of
methylation,
b) mutation
(GCAT
transition).
OH
H
t h ym in e glycol
Adapted from: Zuo S, Boorstein RJ, Teebor GW. (1995) Oxidative
damage to 5-methylcytosine in DNA. Nucl Acids Res. 23:3239-3243.
DNA Oxidation
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Schafer 8
Oxidant Attack on Sugar
The 4’-hydrogen is the weak point
O
O
CH 3
HN
N
O
P
Oxidan t
+
O CH 2
5'
P
1'
3'
P
O
O
4'
O
CH 3
HN
2'
O CH 2
.
N
O
O
P
O
CH 3
HN
O
P
O CH 2
.
H
N
O
+
-
P O
+
Adapted from: C. von Sonntag (1987) The Chemical Basis of Radiation Biology.
Taylor & Francis London, NY.
DNA Oxidation
Society For Free Radical Biology and Medicine
Schafer 9
Transfer of Damage from a Base to a Sugar
O
O
OH
HN
HN
O
O CH2
P
4'
N
.
HO
O
O
P
O CH2
N
H
hydroge n
O
Example: Uracil
radical reacting
with ribose
a b s t r a c t io n
1'
3'
2'OH
O
P
H
OH
P O
O
O
OH
HN
HN
H
H
N
O
P
O
CH2
O
H
P
O
+
P
O
OH
O
CH2
.
P
O
N
O
-
OH
P
(S e e o x id a n t a t t a c k a b o v e )
Adapted from: C. von Sonntag (1987) The Chemical Basis of Radiation Biology.
Taylor & Francis London, NY.
DNA Oxidation
Society For Free Radical Biology and Medicine
Schafer 10
DNA Strand Breaks
Base
Base
P O CH2
4'
O
sugar
O
P O
.
O
sugar
O
sugar
O
-
O P O
O
CH2
P O CH2
N
Base
N
N
-
O P O
O
CH2
O
sugar
Base
N
P O
DNA single strand breaks result because of the collapse of the
sugar. They are the most common damage inflicted by ROS.
DNA Oxidation
Society For Free Radical Biology and Medicine
Schafer 11
Damage by RNS
Peroxynitrite is a strong oxidant formed by
reaction of nitric oxide with superoxide:
•NO
+ O2•-  ONOO- + H+  ONOOH  “•NO2 + HO•“
H
H
N O
O
H
O O
O
N O
N O
O
O
O
Spencer PE. et al. (1996)
Chem Res Toxicol.
9:1152-1158.
DNA Oxidation
N
HN
H2N
N
N
H
OH
+
NO 2
8-Hydroxy-2'-guanine
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Schafer 12
Reaction of Peroxynitrite with
2’-Deoxyguanosine Continued
O
HN
N
H
OH
OH
HN
8-hydroxyguanine
N
N
H2 N
HO
N
H2 N
O
N
N
H
O
ONO
O
HN
N
Adapted from
Douki T. et al. (1996)
Chem Res Toxicol. 9:3-7.
H
H
HO
H
4,5-dihydro-5-hydroxy4-(nitrosooxy)-2'-deoxyguanosine
DNA Oxidation
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H2 N
N
N
H
NO2
8-nitroguanine
Schafer 13
DNA Damage by UV light
dimers:
O
O
O
CH3
HN
CH3
HN
O
N
O
N
NH
N
H
H
O
N
P
S
S
P
S
H3C
HN
hv
O
O
CH3
S
pyrimidine dimers
hydrates:
NH2
NH2
H
N
+
O
Absorption spectra of DNA (calf
thymus) and a protein (BSA) at
equal conc. ( 20 g/mL).
Adapted from: Harm W. (1980)
Biological Effects of Ultraviolet Radiation.
Cambridge University Press.
DNA Oxidation
N
cytosine
hv
N
+ H2O
-H O
2
O
N
O
H
H
OH
H
6-hydroxy-5,6dihydrocytosine
- NH2
-
H2O
H
H N
O
N
H
uracil
Adapted from: Halliwell B, Gutteridge JM. (1989)
Free Radicals in Biology and Medicine
Clarendon Press Oxford 2nd Ed.
Society For Free Radical Biology and Medicine
Schafer 14
Photosensitization can Produce
Singlet Oxygen or Hydroxyl Radical
UVA + Sensitizer
Sen*
1
DNA Oxidation
O2
O2
H2O2
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HO
Schafer 15
DNA-Protein-Crosslinking (DPC):
Produced by UV Light or HO• Attack
O
HN
O
OH
O
CH2
+
Thy
N
HN
Tyr
H
O
DNA
CH2
Protein
CH2
N
H
DNA
OH
O
OH
H
O
CH2
Protein
OH
H
HN
O
-e- - H+
CH2
N
DNA
HN
O
H
CH2
Protein
CH2
N
DNA
H
CH2
Protein
Action spectrum for the
relative induction of DNAprotein crosslinks by UV
and visible radiations
Peak GJ, Peak M J, Sikorski RS,
Jones CA. (1985) Photochem
Photobiol. 41:295-302.
DNA Oxidation
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Schafer 16
O
N
NH
N
HO
1O
2
N
can React with DNA Bases
NH 2
O
H
H
1O
reacts with dG to
form 8 OHdG
OH H
O
2'-deoxyguanosine
N
1O
HO
O
N
O
HO
N
O
2
O
N
N
OH
NH 2
O
H
H
OH H
NH
N
NH
2
NH 2
7,8-dihydro-4-hydroxy-8-oxo2-deoxyguanosine (4R* and 4S*)
1O
2
O
O
H
H
HN
OH H
O
HO
NH
N
O
H
H
N
NH 2
Adapted from: Devasagayam
TPA, Steenken S, Obendorf
MSW, Schulz WA, Sies H.
(1991) Biochemistry. 30:62836289.
OH H
DNA Oxidation
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Schafer 17
Summary
• Oxidants can react with the DNA bases or
sugars
• Guanine is the most sensitive base towards
oxidative attack.
• More then 20 different oxidized base products
are known; some can be mutagenic.
• Damage to the sugar can result in strand
breaks.
• Electron rich moieties are the preferred sites of
attack.
DNA Oxidation
Society For Free Radical Biology and Medicine
Schafer 18