Reactive Oxygen Species and DNA Damage

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Reactive Oxygen Species and DNA Damage

• by
• Manuel H. Constantino

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ROS and DNA Damage

• Overview of ROS
• What are they and where they come from?
• What they do to DNA?
• DNA base products of interaction with ROS
• Repair of Pyrimidine and Purine derived oxidative
  DNA lesions
• Role of ROS Damage in Disease
• ROS endogenous/exogenous scavengers
• Current and future work on ROS

http//www.apsnet.org/education/
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What are ROS and where do they come from?

• O2.-
• Comes from exogenous and endogenous sources.

Andreyev, A.Y., Kushnareva, Y.E. and Starkov,
A.A. Biochemistry (Moscow).2005, 70, 246-264.
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What are ROS and where do they come from?

• H2O2
• SODOX O2.- -gt SODRED O2
• SODRED O2.- 2H -gt SODOX H2O2
• Net 2O2.- 2H -gt H2O2 O2
• OH.
• H2O2 Fe2 -gt OH. Fe3 OH-
• (Fenton reaction)

Keyer, K., Gort, A.S., and Imlay.m J.A. Journal
of Bacteriology. 1995, 177, 6782-6790.
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Where can Oxidative DNA Damage occur in the body?

• In neurons???
• Neurons are likely to suffer from oxidative DNA
  damage than in most other cells.
• The human brain accounts for only 2 of total
  body weight, but 20 of resting oxygen
  consumption due to the high metabolic demand
  required to maintain membrane ion potentials.
• Neurons transcribe about 2-4 times as much DNA as
  do cells from kidney, liver or spleen. Yet
  neurons are non-dividing and must last a
  lifetime.

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What do ROS do to DNA?

• Oxidative attack by OH. on the deoxyribose moiety
  will lead to the release of free bases from DNA.
• ROS generate strand breaks with various sugar
  modifications and production of abasic (AP) sites.

http//www.dojindo.com/newsletter/review_vol2.html
2
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ROS attack on DNA and its Base Products
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DNA Mutation and Damage
200,000 DNA damage events per mammalian cell
per day due to oxidation, hydrolysis, alkylation,
radiation or toxic chemicals. Depurination and
depyrimidation often caused by hydrolysis or
thermal disruption at the AP site. If AP sites
unrepaired, they decay to single-strand
breaks. Pyrimidine dimers are frequently
produced by UV.

• Mutation and DNA Damage.
• Mutation- T __gtA substitution.
• Damage- Adenine loss or G methylation.
• DNA damage tends to interfere with gene
  expression by preventing transcription of RNA
  from DNA.
• DNA mutation usually results in transcription
  that usually produces proteins with diminished or
  altered functionality.
• Mutations that are not lethal to a cell are more
  likely to be perpetuated in dividing cells.

http//www.benbest.com/lifeext/aging.htmlradical
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DNA Mutation and Damage
Types and frequency of DNA damage can be roughly
illustrated by the following table and
representative pictures                         
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DNA Mutation and Damage
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Repair of Pyrimidine and Purine derived Oxidative
DNA lesions

• Oxidized DNA base lesions are removed by
  essentially two types of activity
• Base excision repair (BER)
• Involves removal of single lesions by
  glycosylase action.
• Nucleotide excision repair (NER)
• More complex process involving removal of a
  lesion-containing oligonucleotide.
• Repairs DNA strand damage ranging from 2-30
  bases in length.

• Removal of oxidative DNA lesions is important for
  the limitation of DNA mutations and damage and
  cell cytotoxity.
• Oxidative DNA lesions are subject to multiple,
  overlapping repair processes.

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Repair of Pyrimidine and Purine derived Oxidative
DNA lesions

• BER- primarily repairs damage due to hydrolysis,
  alkylation (usually methylation) or oxidation of
  nucleotide bases.
• Two sub-pathways. Short-patch and long-patch
  pathway.
• The long-patch pathway strips-away 2-10
  nucleotides, including the damaged base.
• Short-patch pathway requires only 3 enzymes
• Uracil-DNA glycosylase.
• AP endonuclease.
• DNA polymerase beta.
• No known diseases associated with inherited
  defects of short-patch BER enzymes.

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Repair of Pyrimidine and Purine derived Oxidative
DNA lesions

• NER repairs cross-links between purines the
  deoxyribose-phosphate backbone due to the
  hydroxyl radical and by pyrimidine dimers caused
  by UV light.
• DNA polymerase delta and DNA polymerase epsilon
  are the specialized DNA polymerases used in NER.
  Many steps and more than 20 proteins are involved
  in unwinding the DNA, in recognizing the type of
  damage to be repaired, etc.
• NER provides backup to BER when glycosylases are
  defective in the nucleus, but NER systems are
  absent from mammalian mitochondria (which only
  have BER).

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Role of ROS Damage in Disease

• A methyl transferase enzyme can repair
  O6-methylguanine by transferring the methyl group
  to its own cysteine.
• The DNA repair enzyme O6-MethylGuanine-DNA
  MethylTransferase (MGMT) is frequently repressed
  by hypermethylation in colon cancer, which
  thereby allows alkylating agents to cause the
  GC-to-AT conversions seen in about half of
  colorectal carcinomas.

A consequence of oxidative base lesions/damage
persisting in DNA is mutation. DNA mutation is a
crucial step in carcinogenesis, and elevated
levels of oxidative DNA damage have been noted in
many tumors, strongly implicating such damage in
the etiology of cancer.
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Role of ROS Damage in Disease
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Role of ROS Damage in Disease
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Role of ROS Damage in Disease

• Almost all Down's syndrome victims have
  Alzheimers Disease by age 50, probably because
  chromosome 21 carries the amyloid gene.
• Chromosome 21 also carries CuZn Superoxide
  Dismutase gene.
• This results in increased production of H2O2,
  which (without catalase or glutathione
  peroxidase) can lead to more hydroxyl radicals.

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ROS Endogenous Scavengers

• Data Collection Resolution 1.5Å X-ray source
  Synchrotron SLS -X10, single wavelength.
• Protein sequence QSVYAFSARPLAGGEPVSLGSLRGKVLLIE
  NVASLGGTTVRDTQMNELQRRLGPRGLVVLGFPCNQFGHQENAKNEEILN
  SLKYVRPGGGFEPNFMLFEKCEVNGAGAHPLFAFLREALPAPSDDATALM
  TDPKLITWSPVCRNDVAWNFEKFLVGPDGPLRRYSRRFQTIDIEPDIEAL
  LSQ

http//www.pdb.org/pdb/explore/images.do?structure
Id2ADQ
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ROS Endogenous Scavengers
Human MnSOD is a tetrameric enzyme with four
identical subunits each harboring a Mn3
atom. M(n1) - SOD O2.- ? M n - SOD O2 Mn
- SOD O2.- 2H ? M(n1) - SOD H2O2. where
M Cu (n1)  Mn (n2)  Fe (n2)  Ni (n2). In
this reaction the oxidation state of the metal
cation oscillates between n and n1.
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ROS Endogenous Scavengers

• Glutathione peroxidase 1 (GPx1)exists in the
  cytoplasm and mitochondria as a homotetramer.
• GPx1 functions as an antioxidant enzyme
  protecting against oxidative stress.
• GPx1 reduces hydrogen peroxide and hydroperoxides
  such as DNA peroxides.

PDB entry 2F8A
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ROS Exogenous Scavengers

• Antioxidants- molecules that slow or prevent the
  oxidation of other chemicals.
• Oxidation is a redox chemical reaction that
  transfers electrons from a substance to an
  oxidizing agent.
• Oxidation reactions can involve the production of
  free radicals, which can form dangerous chain
  reactions.
• Antioxidants- terminate these chain reactions by
  removing radical intermediates and can inhibit
  other oxidation reactions by being oxidized
  themselves.
• As a result, antioxidants are often reducing
  agents such as thiols or phenols.

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ROS Exogenous Scavengers

• BHT
• Vitamin E
• Vitamin A
• Ascorbic acid
• Glutathione

http//employees.csbsju.edu/hjakubowski/classes/ch
331/oxphos/antioxidants.gif
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Current and future work on ROS Prevention to
avoid DNA Damage

• Antioxidants???

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Conclusions

• ROS such as superoxide radical, hydroxyl radical
  and hydrogen peroxide pose a significant threat
  to DNA damage.
• ROS are generated through endogenous and
  exogenous routes.
• Most of the endogenous ROS are produced through
  leaky mitochondria from the electron transport
  chain.
• Exogenous sources are IR, UV, chemotherapeutic
  drugs and macrophages.

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Conclusions

• Oxidative damage produced by ROS results in DNA
  base modifications, single and double strand
  breaks, AP lesions.
• Elevated ROS levels have been implicated in
  disease.
• Elevated levels of 8 OH-G present in diseased and
  cancer cells.
• DNA damage repaired through BER and NER.
• Presence of endogenous and exogenous ROS
  scavengers.

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References

• http//www.apsnet.org/education/
• Andreyev, A.Y., Kushnareva, Y.E. and Starkov,
  A.A. Biochemistry
• (Moscow).2005, 70, 246-264.
• Keyer, K., Gort, A.S., and Imlay.m J.A. Journal
  of Bacteriology. 1995, 177,
• 6782-6790.
• http//www.dojindo.com/newsletter/review_vol2.html
  2
• http//www.benbest.com/lifeext/aging.htmlradical
• http//www.pdb.org/pdb/explore/images.do?structure
  Id2ADQ
• Salmon, T.B., Evert, B.A., Song, B. and Doetsch,
  P.W. Nucleic Acids
• Research. 2006. 32, 3712-3723.
• Lu, H., Zhu, H., Huang, M., Chen, Y., Cai, Y.,
  Miao, Z., Zhang, J., and Ding, J.
• Molecular Pharmacology. 2006. 68, 983-994.
• Feig, D.I., Reid, T.M. and Loeb, L.A. Reactive
  Oxygen Species in
• Tumorigenesis. Cancer Research. 1994. 54,
  1890s-1894s.
• http//employees.csbsju.edu/hjakubowski/classes/ch
  331/oxphos/antioxidant.gif

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• Questions?

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Fe4S4 cluster PDB ID 7ACN
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Repair of Pyrimidine and Purine derived Oxidative
DNA lesions

• NER has two subtypes
• Global-Genome Repair (GGR-gradually covers the
  whole exposed genome and
• Transcription-Coupled Repair (TCR).
• TCR ensures that DNA that is actively being
  transcribed is given the highest priority for
  repair.
• The tumor-suppressor protein BRCA1 (often
  defective in breast cancer) is essential for TCR
  associated with oxidative DNA damage.