DNA Oxidation Society For Free Radical Biology and MedicineSchafer 1 DNA Oxidation: A Simple Overview Freya Q. Schafer, Ph.D. Free Radical and Radiation

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


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


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


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

.


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


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


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


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


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.


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 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


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


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 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


Photosensitization can Produce Singlet Oxygen or Hydroxyl Radical

Sen*

UVA + Sensitizer

1O2 O2 H2O2 HO


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.


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*)


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.

Documents

DNA Oxidation Society For Free Radical Biology and MedicineSchafer 1 DNA Oxidation: A Simple Overview Freya Q. Schafer, Ph.D. Free Radical and Radiation