6: DNA Damage & Repair Flashcards
Give 4 mechanisms of DNA damage, with examples of each
- Chemical cross-linkages (UV)
- Base hydroxylations (Ionising radiation)
- Double/single strand breaks (Ionising radiation -> oxygen free radicals)
- DNA adducts & alkylation (Polycyclic Aromatic Hydrocarbons = benzo[a]pyrene, aflatoxin B1, 2-napthylamine)
What pollutant is formed from combustion of fossil fuels and tobacco?
Polycyclic Aromatic Hydrocarbons
How does Benzo[a]pyrene cause DNA damage?
Forms DNA Adduct by Two step epoxidation of Benzo[a]pyrene
CYP450 forms SECOND epoxide group (extremely reactive)
Forms + charged carbon atom which covalently binds to DNA to form DNA adduct
How does aflatoxin B1 cause DNA damage?
Forms DNA adduct by epoxidation of aflatoxin B1
Aflatoxin is formed by mould (poorly stained grains/peanuts)
Common in Africa/Far east
Liver CYP450 oxidises it to form epoxide (Phase 1 metabolism)
Forms + charged carbon which covalently binds to guanine (DNA adduct)
Potent LIVER carcinogen
How does 2-napthylamine cause DNA damage?
Liver CYP450 metabolises it to form TOXIC metabolite
But quickly inactivated
Acid pH in URINE replaces glucuronide with a positive nitrenium ion which can attack DNA
Hence potent BLADDER carcinogen
How does UV light cause DNA damage?
UV radiation form pyrimidine (thymine) dimers
Example of chemical cross-linkage
How does ionising radiation cause DNA damage?
Generates oxygen free radicals which have unpaired electrons
Free radicals attack electron-rich DNA
Results in Single/Double-strand breaks (double strand much worse)
Leads to apurinic/apyramidinic damage + base hydroxylation (e.g. 8-hydroxy guanine) which is MUTAGENIC
What are the 4 types of DNA repair?
- Direct reversal of DNA damage
- Base excision (for apurinic/apyrmidinic damage)
- Nucleotide excision (for bulky DNA adducts)
- During/post-replication repair
How does Base excision repair work?
- DNA glycosylase - cuts base + adduct from strand
- Endonuclease - cuts DNA strand where base used to be
- DNA polymerase - inserts appropriate base
- DNA ligase - joins strand
How does nucleotide excision repair work?
- Endonuclease cuts strand either side of the adduct
- Helicase unwinds and removes damaged DNA
- DNA polymerase fills in gap with bases
- DNA ligase joins strand
How does p53 deal with DNA damage?
- Cellular stress -> p53 dissociates from MDM2
2. p53 dimerises -> acts as a TF to upregulate genes for DNA repair, growth arrest, senescence or apoptosis
How can unrepaired DNA become a fixed mutation?
Incorrect repair -> DNA replication/cell division = FIXED mutation
Results in either:
- Aberrant proteins
- Carcinogenesis (if oncogenes/TSGs are mutated)
What are the steps involved in testing for potential of a substance to cause DNA damage?
- Structural alerts (chemistry)
- In vitro bacterial gene mutation assay (Ames test)
- In vitro mammalian cell assay (Chromosomal Aberration)
- In vivo mammalian (bone marrow micronucleus)
- Investigative in vivo mammalian assays
What is the Ames test?
- Add rat liver enzyme to convert chemical to active metabolite
- Add active metabolite to bacteria that DON’T synthesise histidine
- Place on histidine-free medium
- If chemical is mutagenic, bacteria will mutate and acquire ability to synthesise histidine and form COLONIES
What is Chromosomal Aberrations test?
- Treat mammalian cell with chemical in presence of liver enzyme
Look for DNA damage under microscope (e.g. chromosome break, chromatid exchange)
