DNA damage and repair Flashcards
Why are the nitrogenous bases so reactive
Carbon ring structures- allowing for potential of delocalisation of electrons
Double bonds- allowing for activation of these
Reactive side chains- just remove methyl group from thymine to make uracil
Generally, planar, cyclical-carbon structures are very reactive, and thus the bases are very reactive too.
What are the potential outcomes of base modifications
Prevent replication
Cause mutations
Describe deamination reactions
The primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to ketogroups
NH2- =O (water in, ammonia out)
Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
Thousands of these occur daily in cells!
Describe chemical modification
The nucleic acid bases are susceptible to numerous modifications by a wide variety of chemical agents. For example, several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism.
All of these can modify DNA bases. A common product of thymine oxidation is thymine glycol:
Describe the consequences of hyper-reactive oxygen species
Hyper-reactive oxygen species are also generated by ionizing radiation (X-rays, gamma rays).
Many environmental chemicals, including “natural” ones (frequently in the food we eat) can modify DNA bases, frequently by addition of a methyl or other alkyl group (alkylation). In addition, normal metabolism frequently leads to alkylation. Addition of larger molecules defines “adducts”.
These thymine glycols can take up these methyl groups and form adducts,
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- bind covalently to DNA
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)
Describe photo damage to DNA
Ultraviolet light is absorbed by the nucleic acid bases, and the resulting influx of energy can induce chemical changes. The most frequent photoproducts are the consequences of bond formation between adjacent pyrimidines within one strand.
Intra-strand effect
Thymine dimers can form
These actually help protect the body from UV damage
Describe the different types of DNA damage that can occur
Nick- break in phosphodiester bond as a result of ionising or radioactive damage
Gap- many nicks
Thymine dimers- disrupts topology of DNA double helix- which can act as a signal for DNA damage
Base pair mismatch- bulge in DNA double helix- no longer have complementary base pairing- can act as a signal for DNA damage.
Describe some of the chemical carcinogens that can damage DNA
dietary lifestyle- smoking environmental occupational medical endogenous
Describe how radiation can damage DNA
Radiation
ionizing
solar
cosmic
Describe the importance of carcinogens
DNA damage can lead to mutation
Mutation may lead to cancer
Damaging DNA is an important strategy in cancer therapy - introduce them into the cells to make them unstable and impossible for them to survive.
State some different types of DNA damage caused by carcinogens
DNA adducts & alkylation Base dimers & chemical cross-links Double & single strand breaks Base hydroxylations & abasic sites formed- hydroxylation damages the base that much it's destroyed.
Which group of chemicals can chemically modify and damage DNA
Polycyclic Aromatic Hydrocarbons which are produced by the combustion of organic material, aflatoxins which are microbial bi-products that contaminate grain crops in hot and humid climates and aromatic amines that are commonly used industrial intermediates. Common environmental pollutants Formed from combustion of fossil fuels Formed from combustion of tobacco
Summarise the mammalian metabolism of carcinogens
§ Phase 1 – addition of functional groups – oxidations, reductions, hydrolysis- takes place in liver- convert hydrophilic carbon ring structures to something that can become more polar and thus excreted.
o Mediated mainly by cytochrome p450 enzymes.
§ Phase 2 – conjugation of phase 1 functional groups – glucuronidation, sulphation, glutathione conjugation, methylation, acetylation & amino-acid conjugation.
o Generates polar (water soluble) metabolites to excrete. Most carcinogens are insidious and only become carcinogenic after phase 1 metabolism.
Where is benzo(a)pyrene BaP found
Coal tar
Cigarette smoke
Grilled meats
Involved in the carcinogenesis of lung and colorectal cancers.
Type 1 carcinogen- very potent
Describe the two step epoxidation 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 and toxic)
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- positively charged
The adducts can joint to chemically modified bases- and thus form covalent bonds with DNA clumping the DNA up
Summarise aflatoxin b1
Formed by Aspergillus flavus mould
Common on poorly stored grains and peanuts
Aflatoxin B1 is a potent human liver carcinogen, especially in Africa and Far-East
Describe the epoxidation of aflatoxin B1
§ Aflatoxin B1 epoxidation process:
o P450 oxidises the aflatoxin B1.
o Aflatoxin B1 2,3-epoxide then adducts to DNA directly using its adjacent N7 positively charged carbon atom.
Adducts on to the N7 position of the guanine-forming a mutation.
Summarise 2-napthylamine
Past components of dye-stuffs
Include 2-naphthylamine and benzidine
Potent human bladder carcinogens
German dye industry epidemiology (1895 Rehn)
Explain the mechanism by which 2-napthylamine is a bladder carcinogen
2-naphthylamine is converted by CYP1A2 to a hydroxylamine derivative, which is reactive (N-hydroxy-2-napthylamine)
In the liver, this is glucuronidated (thus inactivating it)- by gluconoryl 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 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
Describe how UV radiation can cause DNA damage
Pyrimidine (thymine) dimers
Skin cancer
Describe how ionising radiation can cause DNA damage
o Generates free radicals inside cells such as oxygen free radicals – super oxide (O2·) and hydroxyl (HO·).
o These oxygen free radicals have unpaired electrons that are electrophilic and so seek out electron-rich DNA (negatively charged sugar-phosphate backbone of DNA).
Describe the oxygen free radical attack on DNA
§ Double/single stand breaks.
§ Apurinic & apyrimidinic sites – sites where the base is lost whilst the backbone remains.
§ Base modifications:
· Ring-opening – guanine & adenine.
· Glycol (unstable products of oxidation) formation – thymine & cytosine.
· Creation of 8-hydroxyadenine & 8-hydroxyguanine – mutagenic.
Summarise the estimation rates for endogenous DNA damage and repair
Single-strand breaks the most common
Depyrimidation least common
however the max repair rate (BP/hour) per cell is greater than the rate of occurrence of endogenous DNA damage- thus allowing for genomic stability
The greater the persistence of damage then the greater the chance of a mutagenic event
Summarise how a cell responds to DNA damage and cellular stress
Detect cellular insult and then switch on genes to repair the cell. Cellular stresses can include: Oxidative stress Nitric Oxide Hypoxia Ribonucleotide depletion mitotic apparatus dysfunction oncogene activation DNA replication stress Double-strand breaks Telomere erosion
These insults are sensed by the tumour suppressor gene p53
P53 is normally inactivated by a partner protein called MDM2
However, these insults cause MDM2 to be lost, activating p53 and allowing it to act as a transcription factor to turn on gene expression for genes involved in DNA repair
What are the p53 mediated responses to mild and severe physiological stress?
Mild- regulation of p53 mediated genes involved in: metabolic homeostasis antioxidant defence DNA repair growth arrest
Severe stress- p53 mediated protein-protein interactions which lead to apoptosis.
Summarise the different 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
Summarise DNA direct repair
Direct Repair involves the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions
Photolyases repair thymine dimers- this enzyme is activated by visible light (>300nm) absorbed by the chromophore- the enzyme is released and DNA polymerase then restores the native DNA.
O6 methylguanine-DNA methyltransferase (MGMT) directly reverses some simple alkylation adducts or methyl adducts- removes these alkyl and methyl groups at their own expense and are known as ‘suicide’ enzymes.
What is important about DNA mismatches
They are not mutations- they are simply as a result of the error rate of DNA polymerases during replication.
Summarise DNA mismatch repair
Mismatch Repair involves scrutinisation of DNA for apposed bases that do not pair properly.
Mismatches that arise during replication are corrected by comparing the old and new strands [proof-reading].
Preference for newly synthesized strand.
Other systems deal with mismatches generated by base conversions, such as those which result from deamination.
Outline how DNA mismatch repair takes place
MSH and MLH detect mismatch in DNA
nucleases then excise the base and the bases downstream (preference to excise the newly synthesised strand)
DNA polymerases then add the correct bases.
Which base is most electron-rich and hence most capable of attracting electrophiles?
Guanine
Outline base excision repair
mutagen that leads to damage to single base but no damage to backbone can be fixed by only removing base in question (can remove small adducts)
DNA glycosylase hydrolyses between the base and the sugar (forming an apurinic/apyrimidinic site).
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
Re-forms the phosphodiester bonds
outline the nucleotide excision repair pathway
repairing base damage by replacing nucleotides - does damage backbone (can remove larger adducts)- before they form mutations
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- re-forms phosphodiester bond
State 3 human diseases involving defects in nucleotide excision repair
Xeroderma Pigmentosum
Trichothiodystrophy
Cockayne’s syndrome
Multiple genes involved in nucleotide excision repair- so multiple phenotypes exist.
Describe xerdomerma pigmentosum
severe light sensitivity
severe pigmentation irregularities
early onset of skin cancer at high incidence
elevated frequency of other forms of cancer (eye and G.I)
frequent neurological defects
Management focussed at avoiding U.V radiation, ongoing studies analysing whether shifting their activities to the night time will have any improvements.
Summarise trichothiodystrophy
sulphur deficient brittle hair facial abnormalities short stature ichthyosis (fish-like scales on the skin) light sensitivity in some cases
Summarise cockyane’s syndrome
dwarfism light sensitivity in some cases facial and limb abnormalities neurological abnormalities early death due to neurodegeneratio
When do double-strand breaks normally occur
Under physiological conditions during somatic recombination and transposition. e.g. V(D)J recombination
During Homologous Recombination (in meiosis)
As a result of ionizing radiation and oxidative stress induced DNA damage.
Describe homologous DNA double strand repair
Following a double strand break, a 3’-5’ exonuclease exposes a 5’ overhang (essentially a 3’ tail).
Transient base pairing of several nucleotides allows ends to come together
DNA polymerisation (3’ ends act as primers)
Nucleolytic processing (hairpin formed when 3’ tails displace equal strand on another DNA molecule- once it’s been resynthesised- ti re-inserts between the broken strands).
Ligation
DNA double strand equal to the original structure is formed.
Describe non-homologous DNA double strand repair
Ku proteins bind to exposed ends
Recruit DNA-PKcs- which phosphorylates Artemis- ( an nuclease and phosphorylates it) which degrades the ends
Ligase IV and XRC44- joins the broken ends together- forced together- even if they aren’t continuous- you lose the nucleotides removed by the nucleases
Therefore, the duplex that forms is not the same as the original before the break.
Summarise the direct reversal of DNA damage
photolyase splits cyclobutane pyrimidine-dimers
methyltransferases & alkyltransferases remove alkyl groups from bases
Summarise base excision repair
(mainly for apurinic/apyrimidinic damage)
DNA glycosylases & apurinic/apyrimidinic endonucleases + other enzyme partners
A repair polymerase (e.g. DNA Polβ) fills the gap and DNA ligase completes the repair.
Summarise nucleotide excision repair
(mainly for bulky DNA adducts)
Xeroderma pigmentosum proteins (XP proteins) assemble at the damage. A stretch of nucleotides either side of the damage are excised.
Repair polymerases (e.g. DNA Polδ/β) fill the gap and DNA ligase complete the repair.
Summarise during or post-replication repair
mismatch repair
recombinational repair
Outline a schemata for the potential fates of carcinogen-DNA damage
Carcinogen damage leading to altered DNA:
Efficient repair -normal cell
Apoptosis- cell death
Incorrect repair/altered base sequence – DNA replication and cell division: fixed mutations, which can lead to:
Transcription/translation- giving aberrant proteins
Carcinogenesis if critical targets are mutated: oncogenes, tumour suppressor genes.
Summarise the therapeutic agents which cause DNA damage to treat cancer
Agents that induce double strand breaks
Ionising Radiation (Radiotherapy)
Bleomycin
Neocarzinostatin
Agents that make bulky adducts
Cisplatin
Mitomycin C
Alkylating agents Altretamine Bendamustine Bendamustine Hcl Busulfan Carboplatin Uracil Mustard
Try to overwhelm cell with DNA damage such that it has to undergo apoptosis.
outline the hierarchy for assessing the ability of a potential carcinogen/drug to cause DNA damage
Structural alerts/SAR
In vitro BACTERIAL gene mutation assay e.g. Ames test with S. typhimurium
n vitro MAMMALIAN CELL assay
e.g. chromosome aberration,
TK mutation in mouse lymphoma cell
Micronucleus assay
In vivo MAMMALIAN assay
e.g. Bone marrow micronucleus test
transgenic rodent mutation assay
Investigative in vivo MAMMALIAN assays
Describe the bacterial (Ames) test for the 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 (S9- for phase 1 and 2 metabolism) 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 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 in the absence of histidine
the chemical may be converted to a reactive metabolite which damages the DNA and causes mutations.
Describe the in vitro mammalian cell assay for chromosomal aberrations
Treat mammalian cells with chemical in presence of liver S9. Look for chromosomal damage: chromatid exchange chromosome gap double minutes chromosome break acentric ring chromosome interchanges
Describe the in vitro mammalian cell assay for the micronucleus assay
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
Cytochalasin-B holds the cell in the binucleate stage
Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)
Describe the murine micronucleus bone marrow assay
Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei
Bone marrow is pluripotent
The animals are treated with the chemical and their bone marrow cells and 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- so look for presence of micronucelie in polychromatic cells
What is the purpose of the tests that assess DNA damage
The purpose of these tests is to assess the ability of the chemical to damage DNA and thereby determine the risk to humans of the chemical causing mutation or other genomic damage. Since damage to DNA leading to mutation and dysregulation of normal genomic activity in the cell is a feature of cancer, understanding the risks to humans of exposure to chemicals is important.
