DNA Damage & Repair Flashcards
What can damage DNA?
Chemicals
• dietary (40%)
• medical
• lifestyle
Radiation
• ionising
• solar
• cosmic
Types of DNA damage?
DNA double helix
Deamination
Chemical modification
Photodamage
Why is DNA damage so important?
Can lead to mutations
• which can lead to cancer
HENCE
Damaging DNA is an important strategy in cancer therapy
How can carcinogens damage DNA - the 4 mechanisms?
DNA adducts & alkylation
• addition of large carcinogenic groups
Base dimers & chemical cross-links
Base hydroxylation and abasic (base removed) sites form
Double/single strand breaks
Briefly explain mammalian metabolism
2 phases!
Phase 1 - addition of functional groups
• oxidation, reductions, hydrolysis
• mediated mainly by cytochrome p450 enzymes
Phase 2 - conjugations of Phase 1 functional groups
• sulphation, glucuronidation, acetylation, mehtylation
• generates POLAR (water soluble) metabolites to excrete
Fact about carcinogens and metabolism?
Most carcinogens are insidious AND only become carcinogenic AFTER phase 1 metabolism
Explain the metabolism of Polycyclic Aromatic Hydrocarbons
Polycyclic Aromatic Hydrocarbons – common environmental pollutants formed from combustion of fossil fuels or tobacco
2-step B[a]P EPOXIDATION process:
• P450 enzymes oxidise the B[a]P (becomes very reactive)
• EH (epoxide hydrolase) removes the toxic oxide
• P450 again oxidises the B[a]P which then degrades spontaneously
• +VE-charged B[a]P then adducts onto DNA
B[a]P = Benzo[a]pyrene
Explain the metabolism of Aflatoxin B1
EPOXIDATION
Aflatoxin B1:
• formed by Aspergillus flavus mould and is commonly found in poorly stored grains and peanuts
• is a potent liver carcinogen (in Africa/far-east)
Aflatoxin B1 epoxidation process:
• P450 oxidises the aflatoxin B1
• Aflatoxin B1 then adducts to DNA directly using its adjacent N7 positively charged carbon atom.
Explain the metabolism of 2-naphthylamine
2-naphthylamine:
• a past component of dye-stuffs and includes benzidine
• 2-nap is a potent human bladder carcinogen
2-nap metabolism:
• Cytochrome P1A2 oxidises the amine group
• Glucuronyl transferase adds a glucuronide group to the amine which is the broken by the acidic urine pH
• The nitrenium ion remaining then causes DNA damage in the bladder
Explain the metabolism of other carcinogens - solar (UV) radiation?
UV radiation stimulates formation of pyrimidine (thymine) dimers
• causes skin cancer
Explain the metabolism of other carcinogens - ionising radiation
Generates free radicals inside cells such as oxygen free radicals
• super oxide (O2•)
• hydroxyl (HO•)
These oxygen free radicals have unpaired electrons that are electrophilic
• and so seek out electron-rich DNA (negatively charged sugar-phosphate backbone of DNA).
How can oxygen free radicals attack/damage DNA?
Double/single stand breaks
Apurinic & apyrimidinic sites
• sites where the base is lost whilst the backbone remains
Base modifications:
• Ring-opening – guanine & adenine
• Glycol (unstable products of oxidation) formation – thymine & cytosine
• Creation of 8-hydroxyadenine & 8-hydroxyguanine – mutagenic
Relationship between DNA damage and mutation?
The greater the persistence of damage then the greater the chance of a mutagenic event
What enzyme is key for DNA damage repair?
p53
Can sense the DNA damage and regulate the response of the cell to this event
(onenote picture!!)
4 broad types of DNA repair?
Direct reversal of DNA damage – e.g.:
- Photolyase splits cyclobutane pyrimidine dimers (repair thymine dimers)
- Methyltransferases & alkyltransferases remove alkyl groups from the bases (reverse simple alkylation adducts)
Base excision repair – mainly for apurinic & apyrimidinic damage:
- DNA glycosylases & apurinic/apyrimidinic endonucleases
- A repair polymerase (e.g. Pol-beta) fills the gap and DNA ligase completes the repair
Nucleotide excision repair – mainly for bulky DNA adducts:
- Xeroderma pigmentosum proteins (XP proteins) assemble at damage stretch of nucleotides are excised
- A repair polymerase (e.g. Pol-beta) fills the gap and DNA ligase completes the repair
During- or post-replication repair:
- Mismatch repair - compare old and new strand (prefer new strand) and correct it
- Re-combinational repair.
Two type of Exicision repair and enzymes that make up each pathways and are used to repair?
Base excision repair: • DNA-glycosylase – removes the base • AP-endonuclease – cuts the backbone • DNA polymerase – adds a complimentary base • DNA ligase – re-joins the backbone
Nucleotide excision repair:
• Endonuclease – cuts a large swathe of backbone
• Helicase – removes 1+ nucleotides
• DNA polymerase – adds complimentary bases
• DNA ligase – re-joins the backbone
What are the most common damage to DNA that can be seen?
De-purination
&
Single-strand breaks
Fate of Carcinogen-DNA damage (leading to altered DNS)?
Carcinogen damage leading to altered DNA can:
1. Repair
- Apoptosis – If the damage is too much
- Incorrect repair –> DNA replication & cell division (fixed mutation) –> :
a. Transcription/translation to aberrant proteins.
b. Carcinogenesis if critical targets are mutated (e.g. proto-oncogenes and TSGs).
When new drugs are made, they need to be tested to check for their effects on the DNA - what is the order of testing that is carried out?
- SAR
• checking the molecule structurally for groups that could precipitate cancer - Bacterial gene mutation assay (IN VITRO)
• e.g. Ames test w. S.typhimurium - Mammalian cell assay (IN VITRO)
• e.g. chr. aberration - Mammalian assay (IN VIVO)
- Investigate mammalian assays
Explain how Bacterial gene mutation assay isused to test for DNA damage
IN VITRO - Ames
The rat liver enzymes (s9) are used to activate (metabolise) the potential carcinogen so that it becomes potentially toxic
• the bacteria are modified so that they do not produce histidine and so require exogenous histidine to grow and survive
• if the bacteria mutate with the chemical, they can regain the ability to produce histidine and so will grow even without exogenous histidine
Explain how you can detect DNA damage in mammalian cells
Treat mammalian cells with the chemical in the presence of liver s9 enzymes
• inspected directly for chromosomal damage
Explain how you can use Micronucleus Assays to test for DNA damage
IN VITRO
Mammalian cells are treated with the chemical and allowed to divide
• Cytokinesis is blocked using cytochalasin-B
• Bi-nucleate cells are assessed for the presence of micronuclei
The kinetochore proteins are stained to determine if the chemical treatment caused:
• Clastgenicity – chromosomal breakage.
• Aneuploidy – chromosomal loss/gain.
Explain how Bone Marrow Micronucleus tests can be used to test for DNA damage
IN VIVO
Animals are treated with the chemical & the bone marrow cells or peripheral blood cells are examined for micronuclei
• the erythrocytes can usually remove the nucleus during development
BUT
• CANNOT remove the small fragments of DNA (of which the cell forms a micronucleus around).
