Flashcards in DNA Damage and Repair Deck (30)
State some different types of DNA damage caused by carcinogens.
Base dimers and chemical cross-links
Single strand breaks
Double strand breaks
What are abasic sites?
During the repair process, the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from the mutagenic molecule
During replication, this missing base can cause problems
What are the implications of single strand breaks?
These are common and useful
Topoisomerase causes single strand breaks and it is involved in relaxing and unwinding the DNA before replication
What are the implications of double strand breaks?
These are NOT GOOD
The two strands have a tendency to drift apart when a double strand break occurs
There are repair mechanisms for dealing with this, but sometimes the DNA repair can go wrong and introduce DNA damage
What is the usual type of damage that is caused by chemicals?
Why is DNA the target for many carcinogens?
Chemical carcinogens are usually metabolically activated and converted into electrophiles (they want electrons)
DNA is very electron rich
What are the consequences of bulky DNA adducts?
The electrophiles bind and form a covalent bond
The binding of these 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 reaction?
Amino acid conjugation
What are polycyclic aromatic hydrocarbons?
They are environmental pollutants formed from the combustion of fossil fuels and tobacco
Describe the two-step oxidation of benzo[a]pyrene.
B[a]P is a substrate for CYP450, which converts it to B[a]P-7,8-oxide (this is an electrophile)
The body has a defence mechanism – epoxide hydrolase converts the oxide to a dihydrodiol (B[a]P-7,8-dihydrodiol)
This is inactive
However, this dihydrodiol is also a substrate for CYP450, which converts it to another oxide (B[a]P-7,8-dihydrodiol-9,10-oxide)
This even more reactive than the previous oxide – it goes on to form DNA adducts
State two past components of dyestuff that are potent bladder carcinogens.
Benzidine and 2-naphthylamine
Explain the mechanism by which 2-naphthylamine is a bladder carcinogen.
2-naphthylamine is converted by CYP450 to a hydroxylamine derivative, which is reactive
In the liver, this is glucuronidated (thus inactivating it)
The inactive metabolite is excreted by the liver and then it goes to the bladder where it mixes with the urine
The ACIDITY of the urine causes hydrolysis of the glucuronides – this releases the hydroxylamine derivative, which forms a nitrenium ion
This is electrophilic so it leads to the formation of DNA adducts
What does UV radiation lead to the formation of?
Pyrimidine (thymine) dimers – adjacent pyrimidines can covalently link
What does ionising radiation generate?
Name 2 oxygen free radicals.
Superoxide radical (O2.)
Hydroxyl radical (HO.)
What are the consequences of oxygen free radical attack on DNA?
Single and Double strand breaks
Apurinic and apyrimidic sites
Ring-opened guanine and adenine
Thymine and cytosine glycols
8-hydroxyadenine and 8-hydroxyguanine
What are the p53 mediated responses to mild and severe physiological stress?
Mild – repair the damage and restore the 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- and post-replication repair
Give two examples of direct reversal of DNA damage.
Photolyase looks for cyclobutane-pyrimidine dimers and cuts them
Methyltransferases and alkyltransferases remove alkyl groups from the bases
What comes under during and post replication repair?
Which base is most electron-rich and hence most capable of attracting electrophiles?
Describe the process of base excision repair.
DNA glycosylase hydrolyses between the base and the sugar
Then AP endonuclease splits the DNA strand so there is a gap in the backbone
DNA polymerase then fills in the missing base (using the complementary strand as template)
DNA ligase then seals the DNA
Describe the process of nucleotide excision repair.
Endonuclease makes two cuts in the DNA on either side of the site of damage (this demarcates a patch of DNA)
Helicase then removes this patch, leaving the double strand with a patch missing
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 being a normal cell
Severe damage --> apoptosis
Carcinogen causing altered DNA --> incorrect repair/altered primary sequence --> DNA replication and cell division (fixed mutation) --> transcription and 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.
This test usually uses Salmonella typhimurium
The bacterium is genetically engineered so that it can’t produce histidine, so it can only survive and grow on a culture medium that has exogenous histidine
The compound to be tested is, firstly, incubated with rat liver enzymes containing CYP450 enzymes to metabolise the chemical into an active form that can be carcinogenic
The bacteria are mixed with the active chemical and then placed on a culture medium with NO histidine
Any colonies that survive will have become mutated by the chemical so that it regains the ability to produce its own histidine and hence cangrow 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 in the absence of histidine
Describe the use of in vitro micronucleus assays.
This is trying to measure the ability of a chemical to break up DNA into fragments
We need the cell to go through one replication cycle and then stop it when it’s at the binucleus stage – this is when you check for the presence of micronuclei
What is used to block cytokinesis and hold the cell in the binucleate stage in the micronucleus assay?
What are the two types of chromosomal damage that can be detected by this assay?
Clastogenicity – chromosomal breakage
Aneuploidy – chromosomal loss/change in the number of chromosomes