5. DNA damage and repair Flashcards
DNA damage and repair: explain how DNA can be damaged
This is important because DNA damage can lead to mutation and mutation can lead to cancer + Damaging DNA is an important strategy in cancer therapy
Base Modifications Prevent Replication or Cause Mutations
1. Deamination
- The primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to ketogroups in reactions
2. Chemical modification
- Nucleic acid bases = 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
- 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”.
3. Photodamage
- 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.
Outline the types of DNA damage
Types of DNA damage
- Nick: high energy radiation breaks the phosphodiester bond
- Gap: a lot of nicks form a gap where the DNA is no longer two but one stranded
- Dimers: disrupt DNA topology
- Base pair mismatch:
What can damage DNA:
- Chemicals – dietary (40%), medical, lifestyle.
- Radiation – ionising, solar, cosmic.
Damage forms to DNA:
- DNA adducts & alkylation – addition of large carcinogenic groups.
- Base dimers & cross-links.
- Base hydroxylation and abasic (base removed) sites.
- Double/single strand breaks.
Outline types of DNA damage by carcinogens
Base Dimers and Chemical Cross-‐Links
- This is where the DNA molecules are being chemically linked up
Base Hydroxylations
- An oxidative reaction occurring on one of the DNA bases and this can cause problems
- This could mean that the DNA has to get repaired and during the repair process, it could become mutated
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
- During replication, the missing base will cause problems
Single Strand Breaks
- These are very common and can be very useful
- There are physiological enzymes that are responsible for making single strand breaks
- Topoisomerase is involved in the relaxing and unwinding of DNA -‐ it works by chopping the strand of DNA and allowing the strand to unwind and we can gain access to the DNA as the strand is re-‐annealed
- So we can deal with single strand breaks in DNA
Double Strand Breaks
- These are a bit of a disaster
- After the double strand breaks, there is a tendency for the two bits of DNA to drift apart and this is intolerable from the cell’s point of view
- There are a number of DNA repair mechanisms that attempt to amend this, but sometimes the DNA repair can go wrong and introduce DNA damage
DNA Adducts and Alkylation
- This is generally the type of damage that is caused by chemicals
- Some chemicals tend to be metabolically activated into electrophiles (it really wants electrons)
- DNA is very rich in electrons because of all the nitrogens in the bases
- The electrophiles bind to the DNA and form a covalent bond
- The binding of a big bulky chemical to the DNA causes problems particularly during replication because the DNA polymerase runs along the strand and wants to figure out which base to put in next, but it wont be able to do this if it is bound to a big chemical group
- In short, DNA polymerase cannot recognise the base because of the chemical adduct
Outline mechanisms of DNA damage by carcinogens anf the mechanism if mammalian metabolism
Mammalian metabolism
Phase 1 – introduce or unmask functional groups that can be used in Phase 2 – oxidations, reductions, hydrolysis.
Mediated mainly by cytochrome p450 enzymes.
Phase 2 – we use the functional groups (made available by phase 1) to conjugate it with an endogenous molecule to make it water soluble so that it can be excreted in the urine
– glucuronidation, sulphation, glutathione conjugation, methylation, acetylation & amino-acid conjugation.
Generates polar (water soluble) metabolites to excrete.
Most carcinogens are insidious and only become carcinogenic after phase 1 metabolism
- Cyrochrome P450 enzymes (family of 57 enzymes) have a broad substrate specificity and is responsible for oxidising chemicals (very involved in Phase 1 metabolism)
- So the whole purpose of metabolism is to take something that is lipophilic and make it more polar so that we can get rid of it
Polycyclic Aromatic Hydrocarbons (Benzo[a]pyrene)
Polycyclic Aromatic Hydrocarbons
- Common environmental pollutants formed from combustion of fossil fuels or tobacco
Two Step Oxidation of Benzo[a]pyrene (B[a]P)
- P450 enzymes oxidise the B[a]P (becomes very reactive)
- EH (epoxide hydrolase) removes the toxic oxide.
- P450 again oxidises the B[a]P which then degrades spontaneously.
- +ve-charged B[a]P then adducts onto DNA.
This is a model compound that is used to study polycyclic aromatic hydrocarbons
B[a]P is a substrate for CYP450, which oxidises it to form an oxide
(Benzo[a]pyrene-‐7,8-‐oxide)
This oxide is reactive and wants to find electrons (it is an electrophile)
There is a defence mechanism in the body -‐ epoxide hydroxylase cleaves the three membered strained ring of the oxide to form a dihydrodiol (Benza[a]pyrene-‐7,8-‐dihydrodiol) -‐ this is NOT TOXIC
So far, we have converted something that is potentially toxic, to something that is toxic and then detoxified it
THE PROCESS DOES NOT STOP HERE
Unfortunately, the non-‐toxic dihydrodiol metabolite is also a substrate for P450
So P450 converts this non-‐toxic metabolite into another oxide
(benzo[a]pyrene-‐7,8-‐dihydrodiol-‐9,10-‐oxide)
This is very reactive (even more so than the previous reactive oxide) and is desperate to find some electrons to react with
The best source of electrons is DNA so DNA adducts are formed
Epoxidation of Aflatoxin B1
Aflatoxin B1:
- Formed by Aspergillus flavus mould and is commonly found in poorly stored grains and peanuts.
- Is a potent liver carcinogen (in Africa/far-east).
Aflatoxin B1 epoxidation process:
- P450 oxidises the aflatoxin B1.
- Aflatoxin B1 the adducts to DNA directly using its adjacent N7 positively charged carbon atom.
2-naphthylamine
Metabolism of -‐naphthylamine
- 2-‐naphthylamine is a part component of dye-‐stuffs
- Benzidine is another important past component of dye-‐stuffs
- Both benzidine and 2-‐naphthylamine are potent BLADDER carcinogens
NOTE: different targets of different carcinogens
- Polycyclic Aromatic Hydrocarbons cause cancer in many different parts of the body because P450 is involved in its activation and is found in a lot of different tissues
- Aflatoxins primarily target the liver because it is mainly activated by P450 that is found in the liver -Correlated to hepatocellular carcinoma from south east Asia
- 2-‐naphthylamine is a substrate for CYP450, which converts the amino group to form a hydroxylamine (N-‐hydroxy-‐2-‐naphthylamine)
- Hydroxylamines are reactive
- In the liver, when this reactive hydroxylamine is formed, it is glucuronidated (detoxifying reaction) so the chemical is activated and then inactivated
- This glucuronidation is done by glucuronyl transferase
- The inactive metabolite is excreted by the liver and it goes into the bladder and mixes with the urine
- Urine is ACIDIC, and, under acidic conditions, the glucuronides are hydrolysed
- This releases the hydroxylamine derivative, which, in the acidic conditions, rearranges to form a positively charged nitrogen (nitrenium ion)
- The nitrenium ion is an electrophile, which then goes and binds to the DNA and forms adducts
- The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver
Solar radiation
Solar (UV) Radiation
UV radiation can lead to the formation of Pyrimidine Dimers
NOTE:
Pyrimidines = cytosine + uracil + thymine (CUT)
Purines = adenine + guanine (AG)
If there are two pyrimidines next to
each other, under the presence of UV radiation, they can covalently link
The main type of cancer that this causes is
Skin Cancer Ionised Radiation
Skin Cancer Ionised Radiation
Examples of ionising radiation: gamma, X-‐ray, beta particles
These all have the ability to generate chemistry within a cell
They can generate free radicals
Oxygen free radicals are produced by normal biochemistry (e.g. mitochondria can produce oxygen free radicals) and there are good defence mechanisms for dealing with them -‐ however, ionising radiation can overwhelm the defence mechanisms
Super Oxide Radical -‐ very powerful -‐ this is a molecule of oxygen that has an extra electron so it is very reactive
NOTE: if there is an extra electron in the outer orbit, then the molecule will be very reactive
Hydroxyl Radical -‐ a hydroxyl group that has grabbed an extra electron -‐ this is even more reactive than the super oxide radical
These free radicals are very electrophilic and DNA is electron-‐rich
Oxygen Free-‐Radical Attack on DNA
Oxygen Free-‐Radical Attack on DNA
Single strand breaks -‐ not a big deal, we can sort these out
Double strand breaks -‐ very damaging (skin cancer)
Apurinic and apyrimidic sites -‐ base has been oxidised by an oxygen free radical and the DNA repair enzymes come and cut out the base itself, leaving the sugar-‐phosphate backbone in tact so there are gaps (abasic sites)
You can also get base modifications:
Ring-‐opened guanine + adenine
Thymine + cytosine glycols
8-‐hydroxyadenine + 8-‐hydroxyguanine (mutagenic)
Recall the role of p53 in the detection of, and response to DNA damage,
Review of the role of p53 in dealing with cellular stress
- p53 is a crucial tumour suppressor gene
- It is normally tied up with MDM2, which keeps p53 inactive
- P53 is a transcription factor and when MDM2 is lost p53 is activated – enabling dna repair pathways
- When it is released from MDM2, it forms a dimer that activates many pathways
If we have mild physiological stress e.g. DNA repair or growth arrest, p53 orchestrates a transcriptional series of events and activates proteins that help repair the problem
If there is SEVERE stress, then p53 can activate an apoptotic pathway by directly interacting with apoptosis proteins
Many different stresses can kick p53 into activation
Summarise the natural repair mechanisms for damaged DNA, explain how unrepaired DNA may become fixed as a mutation
DNA repair is coded for by more than 100 different genes
- Direct Reversal of DNA Damage
- Photolyase looks specifically for cytlobutane-‐pyrimidine dimers and cuts them
NOTE: solar radiation generates these dimers in the first place
- These photolyase enzymes cut out the dimers and restore the normal sequence
- Methyltransferases and alkyltransferases -‐ remove alkyl groups from bases
REMEMBER: methylation and demethylation is an important way of controlling gene expression
- Sometimes, you can get inappropriate methylation or alkylation and these enzymes will remove these inappropriate groups to restore the DNA structure
Types of DNA Repair
- Direct reversal of DNA damage
- photolyase splits cyclobutane pyrimidine-dimers
- methyltransferases & alkyltransferases remove alkyl groups from bases
- Base excision repair (mainly for apurinic/apyrimidinic damage)
- DNA glycosylases & apurinic/apyrimidinic endonucleases + other enzyme partners
- A repair polymerase (e.g. Polb) fills the gap and DNA ligase completes the repair.
- 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. Pold/b) fill the gap and DNA ligase completes the repair.
- During- or post-replication repair
- mismatch repair
- recombination repair
- These proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis
Excision repair of DNA damage
The most electron-‐rich base is guanine
NOTE: adenine is also very electron rich
If we introduce an electrophile, it will probably target guanine and form a covalent bond -‐ this is toxic and the cell must remove this
Base Excision Repair Pathway
- DNA glycosylase - split/hydrolyses between the sugar and the DNA base (removes base)
- Then an AP-‐endonuclease splits the DNA strand so there is a gap in the backbone
- DNA polymerase then fills in the missing base (adds a complementary base)
- DNA Ligase then seals the DNA to form intact DNA
Nucleotide Excision Repair
- Endonuclease makes two cuts in the DNA on either side of the site of damage
- These patches can be long (100-‐200 nucleotides) or short (~10-‐20 nucleotides)
- Helicase will then remove this patch, leaving the double stranded DNA with a patch missing
- DNA Polymerase then replaces the bases that have been removed using the complementary strand as a template
- DNA Ligase then joins the DNA up again
This process is energy-‐demanding and requires a lot of proteins
Fate of Carcinogen-‐DNA Damage
Estimated rates of endogenous damage and repair
It appears that human cells have plenty of spare capacity to deal with both endogenous and exogenous damage
However, errors begin to creep in, especially with increasing age
If the damage is poorly repaired, then there is greater risk of carcinogenesis
Single strand breaks are the easiest to deal with
Fate of Carcinogen-‐DNA Damage
Carcinogen damage leading to altered DNA can:
- Repair.
- Apoptosis – If the damage is too much.
- Incorrect repair -> DNA replication & cell division (fixed mutation) ->
- Transcription/translation to aberrant proteins.
- Carcinogenesis if critical targets are mutated (e.g. proto-oncogenes and TSGs).
This can lead to transcriptional and translational problems leading to the formation of aberrant proteins or carcinogenesis is critical targets are mutated (e.g. tumour suppressor genes and oncogenes)
Explain how the potential of a chemical / agent to damage DNA is assessed.
Testing for DNA Damage
(in vitro) Bacterial Gene Mutation Assay (Ames):
- The rat liver enzymes are used to activate (metabolise) the potential carcinogen so that it becomes potentially toxic.
- The bacteria are modified so that they do not produce histidine and so require exogenous histidine to grow and survive.
- If the bacteria mutate with the chemical, they can regain the ability to produce histidine and so will grow even without exogenous histidine.
Detecting DNA Damage in Mammalian Cells:
- Mammalian cells with the chemical in the presence of liver S9 enzymes are inspected directly for chromosomal damage.
- Nucleotide excision repair - more than one base wrong
(in vitro) Micronucleus Assays:
- Mammalian cells are treated with the chemical and allowed to divide.
- Cytokinesis is blocked using cytochalasin-B.
- Bi-nucleate cells are assessed for the presence of micronuclei.
- The kinetochore proteins are stained to determine if the chemical treatment caused:
- Clastgenicity – chromosomal breakage.
- Aneuploidy – chromosomal loss/gain.
(in vivo) Bone Marrow Micronucleus Test:
- Animals are treated with the chemical and the bone marrow cells or peripheral blood cells are examined for micronuclei.
- The erythrocytes can usually remove the nucleus during development but CANNOT remove the small fragments of DNA (of which the cell forms a micronucleus around).
Base excision repair - one mistake
