Lecture 3 Flashcards
What affect will loss of DNA repair mechanisms have?
Accelerated mutation rate
Therefore seen in many cancers
What are DNA lesions?
DNA damage which can become mutation when the cell divides if they are not fixed by DNA repair mechanisms
Why do some DNA lesions become mutations?
- Repair is not 100% effective and becomes less precise with age
- Repair pathways can themselves be compromised by mutations to their genes
- Overwhelmed due to number of damaged sites (Massive exposure to carcinogen)
How does could chemotherapy cause recurrence of cancer?
Although chemo is used to kill cancer cells it is itself a carcinogen and thereofre can lead to further mutations which will give rise to new tumours later on
How will the cell normally react to mutations? Why might this not happen?
If severe will cause apoptosis and cell death. Therefore if programmed cell death also becomes defective then a tumour will be born
What are the three main sources of error in DNA?
- Incorporation of errors during DNA synthesis
- Nucleotide Decay
- DNA Damage
What is the chance of DNA polymerase incorpoating an incorrect base? How does the cell decrease this error rate?
1 in 105
Proofreading subunit of subunit will detect the misincorporated nucleotide and cause a reverse of the polymerase to fix the erro; this will decrease incorporation error to 1 in 107
Mismatch repair reduces the error further to 1 in109
How has the importance of the proofreading function been demonstrated?
Mice with knocked out proofreading subunits shown by kaplan-meier plot to have 50% reduction in survival rate to wild type
Oultine mismatch repair
Proteins hMSH2 and hMLH1 detects imperfections in base pairing. Detect which is the template strand and which is replicated strand, using nicks present during replication. (Nicks only on lagging strand it is unknown how direction is determined on leading strand but mismatch repair still successful). Nick tells proteins in which direction to fix to error and a loop is formed between nick and incorrect base, the sequence within the loop is removed and the gap is filled by polymerase.
Where in the genome is mismatch repair known to be most important?
Give an example gene
Regions of short sequence repeats, which are frequently prone to incorrect base incorporations, due to polymerase finding repeats difficult.
TGFß gene has short sequence repeats and commonly mistakes in replication cause premature STOP codon incorporation and a truncated protein product. This will make a cell immune to TGFß and hence cell cycle progression will be unregulated
How has loss of mismatch repair been shown as an early event in cancer development?
Staining of endometrical tissue
Loss of hMLH1 seen in tumour tissue and in surrounding ‘normal’ tissue. Presence in ‘normal tissue’ suggests loss of hMLH1 pre-dates other tumourigenic changes.
What are the three types of nucleotide decay?
Why are they unstoppable?
- Depurination
- Depyrimidination
- Spontaneous Deamination
Natural process that is spontaneous within the cell
Name three types of DNA damage
- Oxidative Damage
- Chemical Damage
- Radiation
What effect does UV light have on DNA?
Formation of pyrimidine dimers - affects ability of double helix to fold and wind appropriately
- Cyclobutane - two rings are connected by two bonds
- 6,4 pyrimidine dimer - rings connected by one bound
How does a cell deal with pyrimidine dimers?
Normally the dimer is repaired however this may not work.
Therefore specialised DNA polymerases, called error prone polymerases, exist that can replicate past the lesion but they can incorporate the wrong base, AKA point muatation. This ‘fixes’ the damage as a mutation in the next generation.
How many lesions per cell exposed to UV per hour?
100,000
Give three examples showing that the location of where the mutagen enters the body is associated with the type of cancer
Polycyclic aromatic hydrocarbons and lung cancer
UV and skin cancer
Aflatoxin and liver cancer
What is aflatoxin? What is its effect?
A product from a fungus on peanuts stored in damp conditions; the toxin then accumulautes in the liver and can cause damage leading to cancer.
What is a DNA adduct?
What is the affect of DNA adducts?
Which agents can cause these?
Damage to a Guanine
Guanine is matched with an A after one round of replication
This A is then matched with a T after the second replication - a GC to TA transition
Polycylic aromatic hydrocarbons, Aflatoxin and heterocylic amines (from cooked meat)
How was the effaicy of smoking on GC to TA transitions shown?
Increased frequency of GC to TA transitions seen in lung tumours patients who smoke over those who do not
What has genome sequencing of 40 types of human tumour shown?
30 ‘signatures’ based of mutation across the genomes
Could be used to determine muations in different types of tumours . However some mutations are common across almost all tumours. Tumours exposed to similar mutagens have some similar signatures.
What role do antioxidants play?
They are converting enzymes which convert mutagens to be non-harmful
How are stem cells protected?
Highly proliferative cells such as stem cells are buried below layers of less-important cells.
How are skin cells protected by UV?
Production of protective melanin
What are the four major pathways to correct DNA damage? How is one different?
- Mismatch Repair
- Nucleotide Excision Repair
- Base Excision Repair
- Double Strand break repair
Mismatch detects ‘normal’ nucleotides that have been mis-incorporated not damaged nucleotides
What does nucleotide excision repair fix?
Pyrimidine dimers
Outline nucleotide excision repair
Complex of 7 proteins that:
- Detect the distortion
- Excise 24 nucleotide on 5’ side and 3 nnucleotides on 3’ side of adduct
- DNA synthesis by special polymerase
- Closure of 3’ gap by ligation
How can Nucleotide excision repair be lost?
What is the outcome?
Mutations in any of the 7 proteins causes loss of nucleotide excision repair
Leads to Xeroderma Pigmentosum - of which 7 variants exist; depending on which protein is mutated. Extreme sensitivity to UV
How is base excision repair different to nucleotide excision repair?
Removal of single base by DNA glycolylase and then rmeoval of sugar and phosphate backbone by APE rather than excision of chain of nucleotides. The gap is then filled by DNA polymerase and ligated like nucleotide excision repair.
How can double strand breaks be triggered?
- Reacitve oxidation species/Ionizing radiation
- DNA tropisomerase inhibitors
- Tropisomerases remove supercoils during replication removes DNA loops. The enzyme creates double strand breaks which they can hold onto or will release if given inhibitors
- Replicative stress
What two pathways repair double strand breaks?
Nonhomologous end joining and Homologous recombination
Outline homologous recombination
When can it occur?
Double strand break is recognised and each strand is excised. Strands will invade into undamaged chromatid; and this chromatid will then be used as template. The strands are the ligated together.
Only during S/G2 phase when another chromatid is available as a template.
Which two genes have been shown to be important in this homologous recombination?
How?
BRCA1 and BRCA2
Loss of the gene leads to breast cancer due to inability to repair double strand breaks
Outline non-homologous end joining
When can this process occur?
Excision either side of double strand break on both strands. Strands brought back together untill limited base pairing possible. Gap filled in and strands ligated.
At any stage of the cell cycle
Which double strand break repair mechanism is better?
Why?
Homologous recombination
Uses undamaged template other sister chromatid to generate accurate repair, whereas, non-homologous end joining leads to loss of sequence.
