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DNA Oxidation Society For Free Radical Biology and Medicine Schafer 1
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: [email protected]
The Virtual Free Radical School
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 2
Components of DNA and RNA
Pyrimidines:
Purines
thymine
HN
NH
O
O
CH3
cytosine
N
NH
O
NH2
5-methylcytosine
N
NH
O
NH2
CH3
uracil
HN
NH
O
O
N
NH
H
N
N
NH2
H
adenine
N
NH
H
HN
N
O
H2N
guanine
bases
sugarsDNA-Structure adapted from:
National Human Genome Research Institute (NHGRI); by artist Darryl Leja http://www.nhgri.nih.gov/DIR/VIP/
HO
OH
OCH2
H
OH
H
H
-D-2-Deoxyribose
HO
OH
OCH2
H
OH
H
OH
-D-Ribose
phosphate back bone
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 3
Hot Spots for Free Radical Attack
-
1'4'3'
2'
NH
N
O
NH2
1
8
76
23
4
5
9
N
NO
O
P O CH2 OO
PO
O
O
O CH2
-
4
53
2 61
CH3
O
O N
HN
1'4'3'
2'
O
OH
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 4
Oxidative Damage to the Bases
Adapted from: von Sonntag C. (1987) The Chemical Basis of Radiation Biology. Taylor & Francis London, NY.
HO• attack on pyrimidinesreducing
oxidizing
123
456
Thymine
HN
NH
O
O
CH3
H+ HO
.
HN
NH
O
O
CH3
H
OH
.
HN
NH
O
O
CH3
HOH
.
HN
NH
O
O
CH2
H
.
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 5
Oxidative Damage to the Bases
Adapted from: von Sonntag C. (1987) The Chemical Basis of Radiation Biology. Taylor & Francis London, NY.
HO• attack on purines
N
NH
N
HN
O
H2N H
Guanine
+ HO.
.N
NH
N
HN
O
H2N H
OH
.N
NH
N
HN
O
H2N HOH
12
3 45
6789
.N
NH
N
HN
O
H2N HHO
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 6
Examples of Oxidized DNA Bases
Adapted from: Dizdaroglu M. (1992) Free Radic Biol Med. 10:225-242. Dizdaroglu M. (2002) Free Radic Biol Med. 32:1102-1115.
N
N
NH2
H
N
N OHH
N
N
O
H2N
HN N
N OHH H
HN
NO
O
H
OH
8-hydroxyadenine 8-hydroxyguanine 5-hydroxyhydantoin
5-hydroxymethyluracil
HN
N
O
O
CH2OH
H H
HN
N
O
O
OH
H
HH
H
HN
N
O
O
OH
5-hydroxy-6-hydrouracil 5-hydroxyuracil
4,6-Diamino-5-formamidopyrimidine
N
N
NH2
NH
NH2
CHO
2,6-Diamino-4-hydroxy-formamidopyrimidine
HN
N
NH
NH2
CHO
O
H2N
8,5'-cyclo-2'-deoxyadenosine
O
NH2
N
N
N
NHO
H
H H
OH H
8,5'-cyclo-2'-deoxyguanosine
O
O
N
NH
N
NHO
H
H H
OH H
NH2
HN
NO
O
CH3
H
H
OH
H
HN
NO
O
CH3
OH
H
OH
HThymine glycol(cis and trans)
HN
NO
O
CH3
H
H
H
H5-hydroxy-6-hydrothymine
5,6-dihydrothymine
Cytosine glycol
HN
N
O
O
OH
OH
H
HN
NO
O
CH3
OHH
N
NO
NH2
OH
H
OH
HH
N
NO
NH2
OH
H5-hydroxycytosine 5,6-dihydroxy
uracil5-hydroxy-5-methylhydantoin
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 7
Oxidation of DNA can lead to Mutations
Misreading of 8-OHdGua can lead mutation (GCAT transversion).
N
N
O
H2N
HN N
N O
H
H
N
N
O
H2N
HN N
N OHH
N
NH
HHN
N
OH2N
NN
O
H
H
HH
H
N
NN
Adenine
8 OH-Gua (enol) 8 oxo-Gua (keto)
Basepairs
A T
G C
8 OHdG A
8 OH-Gua
8-hydroxyguanine
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 8
Oxidation of 5-methylcytosine leads to Rapid Deamination.
Adapted from: Zuo S, Boorstein RJ, Teebor GW. (1995) Oxidative damage to 5-methylcytosine in DNA. Nucl Acids Res. 23:3239-3243.
Deamination can result in: a) loss of methylation, b) mutation (GCAT transition).
N
N
NH2
O
DNA
CH3N
N
NH2
O
DNA
CH3OHH
OH
N
N
O
O
DNA
CH3OHH
OH
H
oxidation
endonuclease III
thymine glycol
5-methylcytosine glycol5-methylcytosine
N
N
NH2
O
H
CH3OHH
OH
N
N
O
O
H
CH3OHH
OH
H
endonuclease III
H2O
NH3
H2O
NH3
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 9
Oxidant Attack on Sugar The 4’-hydrogen is the weak point
Oxidant + 1'2'3'
4'5'
+ P O-
P
HN
N
O
O
CH3
P CH2 OO
O
.P
HN
N
O
O
CH3
CH2 OO
O
P
+
.
HN
N
O
O
CH3
CH2 OO
H
P
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
Transfer of Damage from a Base to a Sugar
Adapted from: C. von Sonntag (1987) The Chemical Basis of Radiation Biology. Taylor & Francis London, NY.
(See oxidant attack above)
P-
+
HO. hydrogen
abstraction
P
1'4'3'
2'
CH2
OH
OO
N
O
HN
O
O
P CH2
OH
OO
N
O
HN
O
OOH
H
H
P
P
CH2
OH
OO
N
O
HN
OH
H
H
HO
O
P
P P
P
.CH2 O
O
N
OOH
HN
O
O
Example: Uracil radical reacting with ribose
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 11
DNA Strand Breaks
DNA single strand breaks result because of the collapse of the sugar. They are the most common damage inflicted by ROS.
.O
4'
Base
sugar
P
O
O O
O
CH2 O N
- -
OCH2 N O
Base
sugarO
CH2 N
Base
sugar
OP
O PP
OP
PO O
O
CH2 O N
Base
sugar
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 12
Damage by RNS
•NO + O2•- ONOO- + H+ ONOOH “•NO2 + HO•“
Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide:
Spencer PE. et al. (1996) Chem Res Toxicol.
9:1152-1158.
N OO O
H
HN
N
O
NH
N
H2NOH NO2
N OO
O
H ON
H
OO
+
8-Hydroxy-2'-guanine
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 13
4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2'-deoxyguanosine
8-nitroguanine
HO
O
H H
HO
H H
N
N NO2N
HN
O
H2N
N
N HN
HN
O
H2N
OH
ONO
N
N OHN
HN
O
H2N H
8-hydroxyguanine
Reaction of Peroxynitrite with 2’-Deoxyguanosine Continued
Adapted from
Douki T. et al. (1996)
Chem Res Toxicol. 9:3-7.
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 14
DNA Damage by UV light
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.
Adapted from: Halliwell B, Gutteridge JM. (1989) Free Radicals in Biology and Medicine Clarendon Press Oxford 2nd Ed.
hydrates:
dimers:
P
hv
pyrimidine dimers
HN
NO
O
CH3
S P
HN
NO
O
S S
O
ON
NH
CH3 H3C
H H
hv+
cytosine
HN
NO
O
CH3
S
N
NO
H
NH2
N
NO
H
NH2
HOH
H
N
NO
H
O
H
H
+ H2O
H2O- H2O
NH2
-
-
uracil6-hydroxy-5,6-dihydrocytosine
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 15
Photosensitization can Produce Singlet Oxygen or Hydroxyl Radical
Sen*
UVA + Sensitizer
1O2 O2 H2O2 HO
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 16
DNA-Protein-Crosslinking (DPC): Produced by UV Light or HO• Attack
+
HN
NO
O
DNA
H
CH2
Tyr
OH
CH2
Protein
OH
CH2
Protein
HN
NO
O
CH2
DNA
H
Thy HN
NO
O
DNA
H
CH2
OH
CH2
Protein
H
CH2
H
DNA
O
O N
HN
OH
CH2
Protein
H
H+-e- -
Action spectrum for the relative induction of DNA-protein crosslinks by UV and visible radiations
Peak GJ, Peak M J, Sikorski RS, Jones CA. (1985) Photochem
Photobiol. 41:295-302.
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 17
1O2 can React with DNA Bases
1O2 reacts with dG to
form 8 OHdG
Adapted from: Devasagayam TPA, Steenken S, Obendorf MSW, Schulz WA, Sies H. (1991) Biochemistry. 30:6283-6289.
O
H H
HO
OH H
N
N
NH
N
O
NH2O O
N
N
NH
N
O
NH2O OH
HOH
O
H H
HO1O2
HN
N
NH
N
O
NH2O
HOH
O
H H
HO
1O2
2'-deoxyguanosine
HOH
O
H H
HO
N
N
NH
N
O
NH2
7,8-dihydro-4-hydroxy-8-oxo-2-deoxyguanosine (4R* and 4S*)
DNA Oxidation Society For Free Radical Biology and Medicine Schafer 18
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.