DNA Damage and repair Flashcards
What can damage DNA?
- Chemicals – dietary (40%), medical, lifestyle. (carcinogens)
- Radiation – ionising, solar, cosmic.
DNA damage can lead to mutation
DNA damaging is used in chemotherapy (they are broken down and unable to function)
- Diet is strongly associated with cancer (about 40-45% of human cancers)
- Herbicides and pesticides contribute to a relatively small proportion of the human cancer burden
- Medical treatments, such as radiotherapy, can also damage DNA and increase the risk of cancer
- Some things are endogenous, for example, mitochondria produce reactive oxygen species that have the ability to damage DNA
- As we grow older we accumulate more and more mutations, which could lead to a neoplastic phenotype
What different forma can DNA damage take?
We’ve talked about the black boxes
Abasic site: the structure is completely, these are the predominant mutations that you see in active cells
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Base Dimers and Chemical Cross-Links
- This is where the DNA molecules are being chemically linked up
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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
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Abasic Sites (based removed site)
- 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
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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 unwinding of DNA
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Double Strand Breaks
- 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 (repair can often lead to mutation)
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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
Review of Mammalian Metabolism
They are all recognised as drugs that need to be removed from the body
- Phase 1 – addition of functional groups – oxidations, reductions, hydrolysis.
- Mediated mainly by cytochrome p450 enzymes.
- Phase 2 – conjugation of phase 1 functional groups – 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**.
Describe how Polycyclic Aromatic Hydrocarbons can cause DNA damage
- Polycyclic aromatic hydrocarbons are common environmental pollutants, formed from combustion of fossil fuels or tobacco
- There is a high connection with colon cancer
- P450 enzymes oxidise the B[a]P (becomes very reactive).
- EH (epoxide hydrolase) removes the toxic oxide. (PRODUCT IS NOT TOXIC)
- P450 again oxidises the B[a]P which then degrades spontaneously.
- +ve-charged B[a]P then adducts onto DNA.
- The best source of electrons is DNA so DNA adducts are formed
This oxide is reactive and wants to find electrons (it is an electrophile)
(EH is a defend mechanism that makes sure that the oxidised B[a]P does not cause any attachment to the DNA)
Describe the Metabolism of 2-naphthylamine and how it can cause cancer
- Both benzidine and 2-naphthylamine are potent BLADDER carcinogens
- Why does the bladder have prevalence for cancer ?
- It is the urine pH in the bladder that makes a very active carcinogen (incapable of causing cancer before)
- 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
- After it is changed, then it is mutated
- 2-naphthylamine:
- A past component of dye-stuffs and includes benzidine.
- 2-nap is a potent human bladder carcinogen.
- 2-nap metabolism:
- Cytochrome P1A2 oxidises the amine group.
- Glucuronyl transferase adds a glucuronide group to the amine (making it less toxic)
- which is the broken by the acidic urine pH.
- The nitrenium ion (positively charged ion) remaining then causes DNA damage in the bladder. (it is an electrophile which results in a DNA adduction)
- The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver
Describe how Solar (UV) Radiation can lead to cancer
- 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 (it stimulates the formation of pyrimidine dimerization)
- The main type of cancer that this causes is Skin Cancer
Describe how Ionised Radiation can lead to cancer
- Examples of ionising radiation: gamma, X-ray, beta particles
- 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
- 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
Describe how free radicals can cause DNA damage
Oxygen Free-Radical Attack on DNA
- Single strand breaks - not a big deal, we can sort these out
- 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 intact so there are gaps (abasic sites)
- You can also get base modifications:
- Ring-opened guanine + adenine
- Thymine + cytosine glycols
- 8-hydroxyadenine + 8-hydroxyguanine (mutagenic)
Review of the role of p53 in dealing with cellular stress
- It is a crucial tumour suppressor gene, responds to a wide variety of insults, kept inactive and at its activation MDM2(keeps it inactive) Is lost, it is a transcriotion factor and can therefore form
- Note and recall that p53 can sense the DNA damage and regulate the response of the cell to this event.
- It is normally tied up with MDM2, which keeps p53 inactive
- 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
What are the different Types of DNA Repair?
DNA repair is coded for by more than 100 different genes
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Direct Reversal of DNA Damage
- Photolyase looks specifically for cytlobutane-pyrimidine dimers and cuts them, recognise the dimers and restore the normal sequence, they are specific for this type of damage
- NOTE: solar radiation generates these dimers in the first place
- 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
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Base Excision Repair - mainly for apurinic/apyrimidic damage
- This is damage where the base has been removed from the DNA
- DNA glycosylases and apurinic/apyrimidic endonucleasesare interested in repairing this damage
- Repair polymerase fill the gap caused by the missing base and DNA ligase repairs it
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Nucleotide Excision Repair - mainly for bulky DNA adducts
- E.g. if polycyclic aromatic hydrocarbons form adducts, the nucleotide excision repair enzymes will try to resolve that
- Xeroderma pigmentosum (XP) proteins assemble at the site of damage - a stretch of nucleotides on either side of the damage are excised
- People that are defective in these enzymes can’t repair the damage from bulky adducts and are prone to developing cancer
- Repair polymerases fill the gap and DNA ligase completes the repair
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During or Post-replication Repair
- Mismatch repair
- Recombinational repair
- These proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis
Describe Mismatch Repair (MMR)
Mismatch repair - repairing erroneous insertion, deletion, and mis-incorporation of normal bases that can arise during DNA replication- failures to maintain normal Watson-Crick base pairing (A•T, C•G)
It can enlist the aid of enzymes involved in both base-excision repair (BER) and nucleotide-excision repair (NER) as well as using enzymes specialized for this function.
*Recognition of a mismatch requires several different proteins including one encoded by MSH2.
*Cutting the mismatch out also requires several proteins, including one encoded by MLH1.
Inactivation of the human mismatch repair system confers a large increase in spontaneous mutability and a strong predisposition to tumor development. Mismatch repair provides several genetic stabilization functions: it corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination. So these genes qualify as tumor suppressor genes.
Question: How does the MMR system know which is the incorrect nucleotide? In E. coli, certain adenines become methylated shortly after the new strand of DNA has been synthesized. The MMR system works more rapidly, and if it detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the correct one and removes the nucleotide on the freshly-synthesized daughter strand. How such recognition occurs in mammals is not yet known.
Describe the Base Excision Repair Pathway
Which base is mostly targeted and why?
- DNA glycosylase split/hydrolyses between the sugar and the DNA base (removes the base)
- Then an AP-endonuclease cuts the backbone
- DNA polymerase then fills in the missing base (it determines the correct base by looking at the complementary strand)
- DNA Ligase then seals the DNA to form intact DNA (re-joins the backbone)
- Mainly for smaller structures
The most electron-rich base is guanine
If we introduce an electrophile, it will probably target guanine and form a covalent bond - this is toxic and the cell must remove this
Describe the process of Nucleotide Excision Repair
To remove the larger guanine
- 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
What is the rate of endogenous damage and repair, which are the most common damages to happen in a DNA molecule?
- The greater the persistence of damage, then the greater the chance of a mutagenic event.
- As it stands, the body has a large capacity for damage with the most common damage to the DNA being:
- De-purination.
- Single-strand breaks.
- 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
What are the different Fates of Carcinogen-DNA Damage?
Carcinogen damage leading to altered DNA can:
- Repair.
- Apoptosis – If the damage is too much.
- Incorrect repair à DNA replication & cell division (fixed mutation, leading to permanent mutations) à:
- Transcription/translation problems which will lead to aberrant proteins.
- Carcinogenesis if critical targets are mutated (e.g. proto-oncogenes and TSGs).
How are new drugs tested for DNA damage?
When new drugs are made, they need to be tested to check for their effect on the DNA. The order of testing is shown to the left.
- SAR – checking the molecule structurally for groups that could precipitate cancer. (structure of the chemical)
- Bacterial gene mutation assay (IN VITRO).
- Mammalian cell assay (IN VITRO). (histones and chromosomes)
- Mammalian assay (IN VIVO). (micronucleus tests in bone marrow, pluripotent cells)
- This means that you can look at the formed elements of the blood as a mechanism of what is happening in the bone marrow and what effect the chemical is having on the bone marrow
- Investigate mammalian assays.
Describe the Bacterial (Ames) Test for Mutagenicity of Chemicals
- 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.
- So, you mix the bacteria with the activated chemical and put them on a plate (which doesn’t have any histidine on it)
- Anything that has NOT been mutated will need exogenous histidine to grow and hence will die on the plate
- The bacteria that have been mutated and regained the capacity to produce histidine will grow and survive
- So, the more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine - this is a very quantitative assay
- This assay also shows a good dose-response relationship
How can you detect DNA Damage in Mammalian Cells? - Chromosomal Aberrations
- We can look at what is happening to the chromosomes themselves
- Chemicals can cause double strand breaks that leads to fragmentation of chromosomes
- This is very labour intensive investigation
Describe In vitro Micronucleus Assays as a way of testing for DNA damage
- Mammalian cells are treated with the chemical in vitro and allowed to divide
- We are trying to measure the ability of the chemical to break up DNA into fragments, then we can count the fragments
- We need the cell to go through one replication cycle and then stop it when a binucleus is formed
- Cytochalasin-B is used to block cytokinesis and hold the cell in the binucleate stage
- Then the binucleate cells are assessed for the presence of micronuclei
- The kinetochores of the chromosomes can be stained to determine if chemical treatment caused:
- Clastogenicity - chromosomal breakage
- Aneuploidy - chromosomal loss/change in the number of chromosomes
- Both clastogenicity and aneuploidy can contribute to cancer
- The cell on the left has NOT been damaged by the chemical
- The cell on the right has extra nuclear material - micronucleus- which is not part of the binuclear and has been generated by the chemical
Describe how Bone Marrow Micronucleus Assay in Mice or Rats can be used for DNA damage
- You are using the pluripotent nature of the bone marrow in producing blood cells
- The animals are treated with the chemical and the bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei
- The erythrocytes normally remove the nucleus during development, but it CANNOT remove small fragments of DNA (e.g. a micronucleus)
- So, if the chemical can generate small fragments of DNA as the erythrocytes are formed from the pluripotent stem cells, these fragments will persist
- The presence of micronuclei in the erythrocytes indicates DNA damage
