6. DNA damage and repair Flashcards
What can cause DNA damage?
Chemicals (carcinogens) • dietary • lifestyle • environmental • occupational • medical • endogenous
Radiation
• ionising
• solar
• cosmic
What percentage of cancer is associated with diet?
40-45%
Do most chemicals damage DNA in their initial form or metabolically converted forms?
Metabolically converted
Give an example of an endogenous cause of cancer
Mitochondria produce reactive oxygen species that may damage DNA
Name 6 ways DNA can be damaged
- Base dimers and chemical-cross-links
- Base hydroxylations
- Abasic sites
- Single strand breaks
- Double strand breaks
- DNA adducts and alkylation
What do base hydroxylations involve?
- Oxidative reaction occurring on one of the DNA bases
- DNA has to be repaired
- Mutation can occur during the repair process
What do abasic sites involve?
- Entire DNA base accidentally removed during the repair process
- Sugar backbone still maintained, but missing base causes problems during replication
What do single strand breaks involve?
• Very common (can be very useful)
• Physiological enzymes usually involved
• Topoisomerase relaxes and unwinds the DNA
- done by chopping the strand of DNA so it can unwind, and gain access as it is re-annealed
• These breaks can therefore be dealt with
What do double strand breaks involve?
- After a double strand breaks, there is a tendency for the 2 bits of DNA to drift apart
- This is intolerable
- Number of DNA repair mechanisms, but sometimes this can go wrong
What do DNA adducts and alkylation involve?
- General type of damage caused by chemicals
- Some chemicals are metabolically activated into electrophiles
- DNA is very rich in electrons (because of nitrogen in bases)
- Electrophiles bind to DNA and form a covalent bond
- DNA polymerase can’t recognise base and work during replication due to this bulk
What is phase I in mammalian metabolism?
- Addition of functional groups (introduce or unmask functional groups
- e.g. oxidations, reductions, hydrolysis
- Mainly cytochrome p450-mediated (oxidation)
What is phase II in mammalian metabolism?
- Conjugation of phase I functional groups
- e.g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation
- generates polar (water soluble) metabolites by adding a polar endogenous group
- easier to excrete
What are polycyclic aromatic hydrocarbons?
- Common environmental pollutants
- Formed from the combustion of fossil fuels and tobacco
- Poisonous and carcinogenic
What is one of the most common polycyclic aromatic hydrocarbons, how is it metabolised and how does it cause damage?
• Benzo[a]pyrene
• Oxidised by CYP450 to produce an epoxide/oxide
- this is reactive and unstable (electrophile)
• Epoxide hydrolase metabolises this into a dihydrodiol
- this is harmless
• Second CYP450 oxidises this to form another oxide (diol epoxide)
- incredibly reactive
- rapidly forms positively derived material (electrophile)
- best source of electrons is DNA
- DNA adducts formed, usually at guanine => mutation
What is aflatoxin B1 and where does it come from?
- Potent human liver carcinogen
- Formed by Aspergillus flavus mould
- Common on poorly stored grains/peanuts
- Especially in Africa and Far-East
Outline the metabolism of Aflatoxin B1 and the way it causes DNA damage
- Oxidised by P450 into B1-2,3-epoxide (very reactive)
- This product reacts with the N7-position of guanine to form bulky DNA adducts
- DNA is now read as damaged
- It’s fixed inappropriately and a mutation has been introduced into the DNA
How can benzo[a]pyrene cause tumours all over the body?
P450 is in cells throughout the body
What is 2-napthylamine and benzidine and where was it common?
- Potent bladder carcinogens
* Used in the German dye industry
Outline the metabolism of 2-napthylamine and how it causes damage
• Substrate for CYP450
- converts the amino group to form a hydroxylamine
• Hydroxylamines are reactive
• They are glucuronidated (detoxified) in the liver by glucuronyl transferase
• Inactive metabolite is excreted and mixes with the urine
- Acidic urine hydrolyses the glucuronides
- Hydroxylamine derivative is released
- Molecule rearranges to form a positively charged nitrogen (nitrenium ion) (electrophile)
- Nitrenium binds to the DNA and forms adducts
- Bladder can’t detoxify the hydroxylamine derivative as the liver
How does solar radiation cause skin cancer?
• UV light leads to the formation of pyrimidine dimers (T, C, U)
- if there are 2 pyrimidines next to each other, UV radiation can cause them to covalently link
• Cell tries to repair this, but introduces a mutation in the process
How does ionising radiation cause cancer?
- All ionising radiation generates free radicals in cells
- This includes oxygen free radicals e.g. superoxide and hydroxyl - very reactive
- Free radicals possess unpaired electrons - seek out electron-rich DNA
Compare the superoxide radical to the hydroxyl radical
- Superoxide radical - molecule of oxygen with an extra electron
- Hydroxyl radical - hydroxyl group that has grabbed an extra electron, even more reactive than superoxide (very electrophilic and DNA is electron-rich)
How do oxygen free radicals damage DNA? (3 ways)
• Single and double strand breaks
- double strand breaks have to be reannealed, can introduce mutatations during the process
- Base can be oxidised by free radical and DNA repair enzymes cut out the base
- Abasic site left
• Base modifications are also introduced:
- e.g. 8-hydroxyadenine + 8-hydroxyguanine (mutagenic)
- while replicating, the machinery has to guess where it is a guanine present, so makes mistakes
Which protein releases p53 for activity during stress on the cell, and what happens to p53 once released?
- Mdm2
- Suppresses activity and keeps it in check
- p53 then forms a dimer that activates many pathways
What does p53 do if there is mild physiological stress e.g. DNA repair or growth arrest?
p53 orchestrates a transcriptional of events and activates proteins that help repair the problem
What does p53 do if there is severe stress?
p53 can activate an apoptotic pathway
What are the 4 types of DNA repair?
1) Direct reversal of DNA damage
2) Base excision repair
3) Nucleotide excision repair
4) During- or post-replication repair
What does direct reversal of DNA damage involve?
- Photolyase splits cyclobutane pyrimidine-dimers formed from UV light to recover the pyrimidines
- Methyltransferases + alkyltransferases remove alkyl groups from DNA bases
What does base excision repair involve?
• Mainly for apurinic/apyrimidinic damage
1) DNA glycosylases hydrolyse between the sugar and affected DNA base, cutting it out
2) Apurinic/apyrimidinic endonucleases split the DNA strand so there is a gap in the SP backbone (can’t just add a base, need the whole nucleotide)
3) A repair DNA polymerase (Polβ) fills the gap formed and fixes it
4) DNA ligase seals the DNA
What does nucleotide excision repair involve?
- Mainly for bulky DNA adducts
- Xeroderma pigmentosum (XP) proteins
1) Endonuclease makes 2 cuts either side of the site of damage
2) Helicase removes this patch
3) Repair DNA polymerases fill the gap using the complementary strand as a template
4) DNA ligase seals the DNA
• Energy-demanding and requires a lot of proteins
What does during-/post-replication repair involve?
- For mismatch repair (for wrong base pairing)
- Also for recombinational repair (recombination occurs in cell replication and can cause big problems)
- Proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis
What is the most electron-rich base?
• Guanine
• Adenosine is also very electron rich
At what level of damage is incorrect repair and altered DNA sequence likely to occur?
Damage that is too high for repair but too low for apoptosis
When a new drug/herbicide etc. is developed, what is the first thing checked for and how can we test if it causes mutations?
- Structure of the chemical - functional groups that could cause problems
- Introduce it to bacteria (Ames test) and see whether it causes mutations
- If it damages the DNA of bacteria, then it has the potential to damage the DNA of mammals
- Then test on mammalian cells (in vitro) - more sophisticated genetic material e.g. histones and chromosomes
- Then test in vivo on mammals using bone marrow micronucleus tests and transgenic rodent mutation assays (very expensive)
Why is bone marrow used to test for the mutagenicity of chemicals?
- Contains pluripotent stem cells that give rise to the cells of the blood
- Can then look at the formed elements of the blood as a mechanism of what’s happening in the bone marrow
- Can also examine bone marrow cells
- It is also a proliferative compartment - expanding the potential to cause a mutation
Outline how the Ames test is carried out
- CYP450 (rat liver enzyme preparation) is added to the bacteria (normally Salmonella typhimurium) - due to poor metabolising activity of bacteria
- This allows the bacteria to convert the chemical into something damaging
- Mixture of enzymes and bacteria are plated
- Bacteria are genetically engineered to not be able to synthesise histidine
- If the chemical causes a mutation to occur, then the bacteria will be able to synthesise histidine
- These bacteria can grow in the absence of exogenous histidine
- Bacteria that haven’t mutated will die without exogenous histidine
- Therefore, the more DNA damaging capability of the chemical, the more colonies grow in histidine absence
How can you check for chromosomal aberrations in mammalian cells when testing chemicals?
- Treat the cells with the chemical in the presence of liver S9
- Then do karyotyping or look at the chromosomal structure
- Can look for chromatid exchange, chromatid gaps etc.
- Difficult, slow and laborious test that requires a lot of technical skill
How is an in vitro micronucleus assay carried out?
- Mammalian cells are treated with the chemical in vitro and allowed to divide
- Trying to measure the ability of the chemical to break up the DNA into fragments, then count the fragments
• Allow the cell to go through one replication cycle then stop it when a binucleus is formed
• Cytochalasin-B is used to block cytokinesis
• Binucleate cells assessed for the presence of micronuclei (pieces of chromosomal material that have been broken off and no longer appear in the nucleus)
• The kinetochores can be stained to determine if chemical treatment caused clastogenicity or aneuploidy
- both contribute to cancer
• Normal cell with appear to have 2 nuclei, damaged cell will appear to have an extra micronucleus
What is clastogenicity and aneuploidy?
- Clastogenicity - chromosomal breakage
* Aneuploidy - chromosomal loss / change in the number of chromosomes
How can you tell if a rat has been affected by chemical treatment of it’s bone marrow?
Erythrocytes cannot remove small fragments of DNA e.g. micronucleus, so they will be present
