DNA Damage and Repair Flashcards
Describe the different base modifications that prevent replication or cause mutations
Spontaneous deamination:
- Removal of the somewhat unstable primary amino group of nucleic acid bases => ketogroups
e. g. cytosine to uracil, adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
Chemical damage:
- Hyper-reactive oxygen species generated by ionising radiation or as byproducts during normal oxidative metabolism can modify DNA bases, e.g. thymine to thymine glycol
- Many environmental chemicals can modify DNA bases by addition of methyl/alkyl groups (alkylation); addition of larger molecules defines ‘adducts’
Photodamage:
- UV light induces covalent bond formation between adjacent pyrimidines within one strand => pyrimidine dimer
Summarise the different causes of DNA damage
Chemicals (carcinogens): dietary lifestyle environmental occupational medical endogenous
Radiation:
ionizing
solar
cosmic
State some different types of DNA damage caused by carcinogens.
Base dimers and chemical cross-links Base hydroxylations Abasic sites Single strand breaks Double strand breaks DNA adducts
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
Why is DNA the target for many carcinogens?
Chemical carcinogens are usually metabolically activated and converted into electrophiles;
DNA is very electron rich (many double bonds, ring structure)
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
Give a brief overview of phase 1 and phase 2 metabolism
Phase I
- addition of functional groups via e.g. oxidations, reductions, hydrolysis
- mainly cytochrome p450-mediated
Phase II
- conjugation of Phase I functional groups
e. g. Glucuronidation, Acetylation, Sulphation, Methylation, Amino acid conjugation, Glutathione conjugation - Generates polar (water soluble) metabolites.
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. How does lead to DNA damage?
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
Where does aflatoxin B1 come from? What type of carcinogen is it, and it which countries do you find the greatest cancer incidences?
- Formed by Aspergillus flavus mould which is common on poorly stored grains and peanuts
- It is a potent human liver carcinogen, especially in Africa and Far-East
Describe the metabolism/epoxidation of aflatoxin B1
Converted to aflatoxin B1, 2,3-epoxide by CYP450.
This metabolite then reacts with the guanine bases at the N7 position => DNA adduct formation
State two past components of dyestuffs that are potent carcinogens. State also the type of carcinogen
Benzidine and 2-naphthylamine
Potent human bladder carcinogens
Describe and explain the mechanism by which 2-naphthylamine is metabolised to become a bladder carcinogen.
- 2-naphthylamine is converted by CYP450 to a reactive hydroxylamine derivative
- In phase II (also in liver) it is then inactivated by glucuronidation (catalysed by glucuronyl transferase)
- 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 => hydrolysis of the glucuronides and subsequent formation of a nitrenium ion
=> This is electrophilic so it leads to the formation of DNA adducts
What does UV radiation lead to the formation of in DNA?
Pyrimidine (thymine) dimers
What does ionising radiation generate?
Free radicals in cells
Name 2 oxygen free radicals. What makes them so dangerous?
Superoxide radical (O2.) Hydroxyl radical (HO.)
They possess unpaired electrons so are electrophilic and therefore seek out electron-rich DNA
What are the consequences of oxygen free radical attack on DNA?
- Single and Double strand breaks
- Apurinic and apyrimidic sites
Base modifications:
- Ring-opened guanine and adenine
- Thymine and cytosine glycols
- 8-hydroxyadenine and 8-hydroxyguanine (mutagenic)
What is the p53 gene?
The p53 gene lies on chromosome 17 and codes for a nuclear phosphoprotein having three major roles:
- It can activate DNA repair proteins when DNA has sustained damage
- It can arrest growth by holding the cell cycle at the G1/S checkpoint allowing time for repair proteins to fix the damage
- Participation in initiating apoptosis.
What are the p53 mediated responses to mild/physiological and severe cellular stress (described earlier)?
Mild – repair the damage and restore the normal function of the cell
Severe – apoptosis
What are the main mechanisms of DNA repair?
Direct reversal of DNA damage
Base excision repair
Nucleotide excision repair
During- and post-replication repair
Describe two examples of direct reversal of DNA damage.
- Photolyase splits cyclobutane-pyrimidine dimers
2. O6 methylguanine-DNA methyltransferase or MGMT (and alkyltransferases) remove alkyl groups from the bases
Describe the process of base excision repair (BER)
DNA glycosylase hydrolyses between the base and the sugar (removes base and adduct)
AP endonuclease causes a break in the DNA backbone (where the removed base was)
DNA polymerase then fills in the missing base (using the complementary strand as template)
DNA ligase then fixes the DNA backbone
Describe the process of nucleotide excision repair (NER)
Endonuclease makes two cuts in the DNA on either side of the site of damage (this demarcates a patch of DNA on the one strand)
Helicase then removes this patch
DNA polymerase replaces the missing bases
DNA ligase joins the DNA up
What comes under during and post replication repair?
Mismatch repair
Recombinational repair
