DNA Damage and Repair 2 Flashcards
What can carcinogens do to DNA
Form abasic sites: base absent though ribophosphate backbone remains
Form base dimers: between adjacent bases on the same strand
Form DNA adducts: covalent binding of other compounds to DNA) including alkylation
Hydroxylate bases: meaning they are no longer recognised/functional
Single or double strand breaks: single strand breaks can be repaired easily, doubles can’t)
Give an example of a family of carcinogens and what they are formed from
Polycyclic aromatic hydrocarbons
Common environmental pollutants formed from the combustion of fossil fuels or tobacco
Describe benzo[a]pyrene
Insoluble in water, and to be excreted in urine it needs to be, so it undergoes metabolism
Describe the metabolism of benzo[a]pyrene
- Oxidation by cytochrome P450 oxidase, giving it a reactive epoxide ring
- Epoxide hydrolase converts it to a diol
- Cytochrome P450 adds another epoxide
What is the effect of the metabolism of benzo[a]pyrene
Makes benzo[a]pyrene soluble, but also incredibly electrophilic. The best source of electrons in the cell is DNA (or protein), so b[a]p ends up forming adducts to bases, especially guanine.
Where is aflatoxin B1 found
food stores contaminated with aspergillus
Describe the metabolism of aflatoxin B1
epoxide added by a P450 and can adduct to guanine
Describe the metabolism of 2-napthylamine
- Amine group hydroxylated to an amide by P450 (phase I)
- Conjugated to glucoronide via glucoronyl transferase (Phase II) in the liver
- This allows it to enter the urine
- Acidic pH of urine causes the dissociation of the bond between the 2-napthylamine and the glucoronide
- This leaves an electrophilic nitrenium ion, which finds electrons in the DNA of bladder cells
Where is 2-npahtylamine found
Common in dyes in the past
What is deamination and give an example
Primary amino groups of nucleic acid bases can be converted to ketogroups
cytosine conversion to uracil
What is chemical modification
Nucleic acid bases are susceptible to modifications by a wide variety of chemical agents
Common for Adduct formation
Several types of hyper-reactive oxygen can modify DNA bases
What is a common product of thymine oxidation
Thymine glycol
How can hyper-reactive oxygen species be generated
As byproducts during normal oxidative metabolism Ionising radiation (x-rays, gamma rays)
How do environmental chemicals modify DNA bases
Addition of methyl or alkyl groups
What carcinogens can cause to damage DNA
dietary lifestyle environmental occupational medical endogenous
Which types of radiation can damage DNA
ionizing (UV, x-ray, or gamma)
solar
cosmic
What is photodamage
Ultraviolet light is absorbed by the nucleic acid bases
The resulting influx of energy can induce chemical changes
What are the most frequent photoproducts consequences of
Bond formation between adjacent pyrimidines within one strand.
Explain how ionising radiation is able to damage DNA
Generates free radicals in cells (most often from water)
super oxide radical (O2•)
hydroxyl radical (HO•)
These possess unpaired electrons, and so are electrophilic. They damage DNA very easily
Give examples of how free radicals can modify bases
- forming pyrimidine glycols
- opening the rings of purines
- hydroxylating purines to 8-hydroxyguanine (the most mutagenic) or 8-hydroxyadenine
Give examples of how free radicals (e.g. from oxygen) affect DNA (base modifications, free radicals, UV)
Base modifications can lead to abasic sites as the defective base is removed.
Free radicals can also cause single or double strand breaks.
UV can cause base dimers to form between pyrimidines (a bit different, as not all UV is ionising).
What is the role of p53 in dealing with cellular stress
(tumour suppressor gene) Responds to cellular insults: Mitotic apparatus dysfunction DNA replication stress Double-Strand breaks
What is the regulation mechanisms of p53
MDM2 keeps p53 inactive.
It is then lost in damage, allowing p53 to activate transcriptional pathways, including DNA repair
What are the types of DNA repair
Direct reversal of DNA damage
Base excision repair (mainly for apurinic/apyrimidinic damage)
Nucleotide excision repair (mainly for bulky DNA adducts)
During or post replication repair
Describe direct DNA repair
the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions
Give an example of direct DNA repair
photolyase splits pyrimidine dimers (opposite of UV action), repairing thymine dimers
methyltransferases / alkyltransferases remove alkyl groups from DNA
Describe base excision repair of DNA damage
Damage to base (but not to the phosphodiester backbone)
- DNA glycosylase removes the base of the nucleotide without affecting the backbone
- AP-endonuclease cuts the DNA strand open
- DNA polymerase adds the correct base (e.g. Pol-beta)
- DNA ligase closes the strand
Describe the nucleotide excision repair of DNA damage
- Endonuclease removes the phosphodiester bond
- Helicase removes a large chunk of bases (including backbone)
- DNA polymerase remakes the strand
- DNA ligase closes strand
Give examples of human genetic disease involving nucleotide excision repair
Xeroderma pigmentosum
Trichothiodystrophy
Cockayne’s syndrome
Describe DNA double strand break repair
Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating.
Non-complementary nucleotides - Nonhomologous End-Joining (NHEJ).
What are the types of therapeutic agents used to cause DNA damage in tumour cells
Alkylating agents
Agents that make bulky adducts
Agents that induce double strand breaks
Explain how is DNA damage tested for
- Structural alerts/SAR
- In vitro bacterial gene mutation assay
- in vitro mammalian cell assay
- in vivo mammalian assay
- Investigation in vivo mammalian assays
Describe the Ames test for mutagenicity of chemicals
- Bacteria that do not synthesis histidine (AA) are bathed in the chemical to be tested and a enzyme preparation
- Placement on histidine-free media
3, If a colony forms, then its means the bacteria has mutated and acquired the ability to synthesise histidine - Counting the number of colonies quantifies the mutagenic capability of the chemical.
Give an example of a bacterium that does not synthesise histidine
Salmonella
