DNA Damage and Repair Flashcards
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
Describe the use of in vitro micronucleus assays.
Attempts to measure ability of a chemical to break up DNA into fragments
Let cell to go through 1 replication cycle + then stop it at the binucleus stage- here check for the presence of micronuclei
What is used to block cytokinesis and hold the cell in the binucleate stage in the micronucleus assay?
Cytochalasin-B
What are the two types of chromosomal damage that can be detected by an in vitro micronucleus assay?
Clastogenicity: chromosomal breakage
Aneuploidy: chromosomal loss/change in number of chromosomes
Explain the reasoning behind the use of bone marrow micronucleus assays to test the mutagenicity of a chemical.
Bone marrow is pluripotent
Animals are treated with the chemical + their bone marrow cells + peripheral erythrocytes are examined for the presence of micronuclei
Erythrocytes normally remove the nucleus during development, but it CANNOT remove small fragments of DNA e.g. a micronucleus
So the presence of micronuclei in erythrocytes indicates DNA damage
List 3 types of base modification that can prevent replication/ cause mutations
Deamination e.g. Cytosine to Uracil
Chemical modification e.g. oxidation of thymine to thymine glycol, addition of alkyl/ methyl groups
Photodamage: UV influx of energy causes intrastrand changes e.g. Thymine dimer formation
How do different types of DNA damage present in a DNA strand?
Base mismatch (mutated base) = Bulge Thymine dimers = Distortion in helix Radiation causing a break in phosphodiester backbone = Nick Multiple nicks adjacent to each other = Gap (single strand DNA with opposite strand missing)
What can damage DNA?
Carcinogens (Dietary, Lifestyle, Environmental, Occupational, Medical, Endogenous)
Radiation (Ionising, Solar, Cosmic)
What are the main metabolism phase I reactions and what mediates these?
Oxidations, Reductions, Hydrolysis
Cytochrome p450 mediated
How does aflaxtoxin B1 become carcinogenic?
Converted by CYP450 to an epoxide (very active)
Epoxide reacts with guanines, results in adduct formation causing mutations
In what state is p53 found? When does this change?
Kept inactive by MDM2
Cell insult causes MDM2 to be lost + activation of p53
p53 is a transcription factor able to activate DNA repair pathways
Describe DNA mismatch repair
Bulge in replication fork indicates error made by DNA polymerase
Recognised by MSH + MLH proteins which bind around the mismatch + cut out offending + surrounding bases
DNA polymerase restores correct sequence
Give an example of a disease caused by a mutation in nucleotide excision repair pathway
Xeroderma Pigmentosum
Describe the process of homologous double strand break repair
2 fragments with mismatching ends
Enzyme cuts back ends to reveal more single stranded DNA in hope it will find sequences that match each other + repair
Describe the process of non-homologous double strand break repair
Free DNA ends picked up by KU proteins
KU proteins hold DNA ends together, forcing them to join
Give 3 examples of therapeutic agents that cause DNA damage
Alkylating agents: cause adduct formation, overwhelms cell causing apoptosis
Agents that make bulky adducts e.g. Cisplatin
Agents that induce double strand breaks e.g. ionising radiation
