DNA Damage and Repair Flashcards
What is deamination and give examples
Primary amino groups of nucleic acid bases can be converted to ketogroups (C->U)
Adenine -> hypoxanthine
Guanine -> xanthine
5-methyl cytosine -> thymine
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 (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) 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 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.
What are the types of DNA damage
Nick
Gap
Thymine dimer
Base pair mismatch
What carcinogens can cause to damage DNA
dietary lifestyle environmental occupational medical endogenous
Which types of radiation can damage DNA
ionizing
solar
cosmic
Describe the DNA damage by carcinogens
DNA adducts and alkylation
Base hydroxylations
Base dimers and chemical cross-links
Double and single strand breaks
Describe the phases of metabolism
Phase I - addition of functional groups
Mainly cytochrome P450-mediated
Phase II - Conjugation of phase I functional groups Generates polar (water soluble) metabolites
Give examples of Phase I reactions
Oxidation
Reduction
Hydrolysis
Give examples of phase II reactions
Sulphation Glucuronidation Acetylation Methylation Amino acid Glutathione conjugation
Where can polycyclic aromatic hydrocarbons, aflatoxin B1 and 2-napthylamine be found
Polycyclic hydrocarbons - Formed from combustion of fossil fuels and tobacco
Aflatoxin B1 - grains and peanuts
2-napthylamine - Dye-stuffs (past)
Describe the epoxidation of B[a]P
2 steps
B[a]P is converted to DNA adducts using liver P450 and epoxide hydrolase
DNA adducts attaches itself to bases and causes mutations
Describe the epoxidation of aflatoxin B1
Conversion first by cytochrome p450 oxidase into a very active form.
Reaction with guanine on DNA (N7 position) to cause adduct formation
Describe the metabolism of 2-naphthylamine
First conversion by cytochrome p450 oxidase (CYP1A2)
Second is a phase II reaction using glucuronyl transferase
pH of the urine causes conversion to a DNA-reactive electrophile that causes bladder tumours
What is an adduct
Segment of DNA bound to a cancer-causing chemical
Often from the conversion of a small molecule to a much larger one
Explain how UV radiation is able to damage DNA
Pyrimidine (thymine) dimers
Skin cancer
Explain how ionising radiation is able to damage DNA
Generates free radicals in cells
Includes oxygen free radicals
super oxide radical (O2•)
hydroxyl radical (HO•)
Possess unpaired electrons
electrophilic and therefore seek out electron-rich DNA
Describe the oxygen free radical attack on DNA
Double and single strand breaks
Apurinic + apyrimidinic sites (reactive)
Base modifications
What are the base modifications that oxygen free radicals can cause
ring-opened guanine +adenine
thymine + cytosine glycols
8-hydroxyadenine + 8-hydroxyguanine (mutagenic)
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
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
What is the actions of MGMT and photolyases
directly reverses some simple alkylation adducts
Photolyases repair thymine dimers
How are DNA mismatches repaired
Involves scrutinisation of DNA for apposed bases that do not pair properly
Mismatches that arise in replication are corrected via proof-reading (comparison of old and new) - preference for the new strand
Other systems deal with mismatches generated by base conversions, such as those which result from deamination
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
- 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
When are double-strand breaks made
Under physiological conditions during somatic recombination and transposition. e.g. V(D)J recombination
During Homologous Recombination.
As a result of ionizing radiation and oxidative stress induced DNA damage.
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 consequences of incorrectly repaired carcinogen damage leading to altered DNA
DNA replication and cell division -> fixed mutation
Transcription/translational gives aberrant proteins
Carcinogenesis if the targets are mutated
Apoptosis and cell death
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
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
The chemical tested is added to rate liver enzyme preparations (S9) and bacteria that do not synthesise histidine
Conversion of chemical to reactive metabolite
On histidine-free media: if mutations occur in bacterial genome
then bacteria acquire ability to synthesise histidine = colonies
Give an example of a bacterium that does not synthesise histidine
Salmonella
How are chromosomal aberrations detected
Treat mammalian cells with chemical in presence of liver S9. Look for chromosomal damage
Describe the in vitro micronucleus assay
Cells treated with chemical and allowed to divide
Binucleate cells assessed for presence of micronuclei
Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)
Describe the murine bone marrow micronucleus assay
Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei
