DNA Damage and Repair Flashcards
(39 cards)
State 5 different types of DNA damage caused by carcinogens.
Base dimers + chemical cross-links Base hydroxylations + Abasic site formation Single strand breaks Double strand breaks DNA adducts + alkylation
What are abasic sites?
Result from base hydroxylation
Base itself is sufficiently changed that there is no longer a base there
Results in a base on one strand having no partner on the other strand
What is the usual type of damage that is caused by chemicals? What are these?
DNA adducts
Bases covalently linked to other chemical entities
Why is DNA the target for many carcinogens?
Carcinogens usually metabolically activated + converted into electrophiles (want electrons)
DNA is very electron rich
What are the consequences of bulky DNA adducts?
Electrophiles bind + form a covalent bond
Binding of adducts causes problems, particularly during replication because it interferes with the ability of DNA polymerase to recognise the base (because of the bulky adduct)
What are the six types of Phase II metabolism reactions?
Glucuronidation Acetylation Sulphation Methylation Amino acid conjugation Glutathione conjugation
What are polycyclic aromatic hydrocarbons?
Environmental pollutants formed from the combustion of fossil fuels + tobacco
Describe the two-step oxidation of benzo[a]pyrene.
CYP450 converts B[a]P to B[a]P-7,8-oxide
The body has a defence mechanism: Epoxide hydrolase; converts the oxide to a dihydrodiol (inactive)
This dihydrodiol is also a substrate for CYP450, which converts it to another oxide which is even more reactive than the previous: goes on to form DNA adducts
State two past components of dyestuff that are potent bladder carcinogens.
Benzidine
2-Naphthylamine
Explain the mechanism by which 2-naphthylamine is a bladder carcinogen.
CYP450 converts 2-naphthylamine to a reactive hydroxylamine derivative
Glucuronidated in the liver, (inactivating it)
In bladder it mixes with urine
ACIDITY of urine causes hydrolysis of the glucuronides; this releases the hydroxylamine derivative, which forms a nitrenium ion
This is electrophilic so leads to formation of DNA adducts
What does UV radiation lead to the formation of?
Pyrimidine (thymine) dimers: adjacent pyrimidines covalently link
Drive skin cancer
What does ionising radiation generate?
Free radicals in cells
Name 2 oxygen free radicals. What is the consequence of them?
Superoxide radical (O2.) Hydroxyl radical (HO.) Possess unpaired electrons, thus electrophilic + seek electron rich DNA
What are the consequences of oxygen free radical attack on DNA?
Single + Double strand breaks Apurinic + apyrimidic sites Base modifications: Ring-opened guanine + adenine Thymine + cytosine glycols 8-hydroxyadenine + 8-hydroxyguanine
What are the p53 mediated responses to mild and severe physiological stress?
Mild: repair damage + restore normal function of the cell
Severe: apoptosis
What are the main types of DNA repair?
Direct reversal of DNA damage
Base excision repair
Nucleotide excision repair
During- + post-replication repair
Give two examples of direct reversal of DNA damage.
Photolyase looks for thymine dimers + cuts them out (repaired by DNA polymerase)
Methyltransferases + alkyltransferases remove alkyl groups from the bases
What comes under during and post replication repair?
Mismatch repair
Recombinational repair
Which base is most electron-rich and hence most capable of attracting electrophiles?
Guanine
Describe the process of base excision repair.
DNA glycosylase hydrolyses between the base + the sugar
Then AP endonuclease splits the DNA strand so there is a gap in the backbone
DNA polymerase fills in the missing base (using the complementary strand as template)
DNA ligase seals the phosphodiester backbone
Describe the process of nucleotide excision repair.
Endonuclease makes 2 cuts in the DNA on either side of the site of damage (demarcates a patch of DNA)
Leaving a gap
DNA polymerase replaces the missing bases
DNA ligase joins the DNA up
Describe the possible fates of carcinogen-DNA damage.
Low level of damage= effective repair: return to normal cell
Severe damage= apoptosis
Incorrect repair/altered primary sequence= DNA replication + cell division (fixed mutation): transcription + translation giving aberrant proteins + carcinogenesis if critical targets are mutated
Describe the process of testing whether a chemical can cause carcinogenesis.
Look at structure of compound
Test in vitro on bacteria
Test in vitro on mammalian cells
Test in vivo on mammals
Describe the bacterial (Ames) test for mutagenicity of chemicals.
Bacterium GM so it can’t produce histidine, thus only survives + grows on a medium that has exogenous histidine
Compound to be tested is incubated with rat liver enzymes containing CYP450 enzymes to metabolise the chemical into an active form that can be carcinogenic
Bacteria are mixed with the active chemical + then placed on a medium with NO histidine
Any colonies that survive must have been mutated by the chemical so it regains the ability to produce its own histidine + hence can grow in the absence of histidine
Any bacteria that hasn’t been mutated will die on the dish
The greater the DNA damaging capability of the chemical, the more colonies will grow
