Genome instability and checkpoints Flashcards
In intestinal epithelia what is responsible for
1) LOH at Chr5. 5q
2) Formation of early adenomas from hyperplastic epithelium
3) LOH of Chr 18q
4) LOH of Chr 17p
1) Due to loss of APC
2) Due to DNA hypomethylation
3) Due to a loss of SMAD4
4) Due to a loss of p53
How are the mutations in tumourous tissue compared to adjacent normal tissue
Using a SNP array and comparitive genomic hybridisation to identify regions which contain genomic imbalance, LOH and copy number variants.
In adenocarcinomas, how many mutations are there per cell roughly, and how many of these are drivers of cancer development
around 80 mutations with around 15 drivers
What is the mutator hypothesis
Tumour cells acquire a mutator gene that increases the rate of mutation as an early event in the cancers development
What two forms of instability are there as a result of early mutations in cancerous cells
CIN - Chromosomal instability
MIN - Microsatellite instability
What results from chromosomal instability
Chromosomal aberrations- rearrangements
LOH
aneuploidy/ polyploidy
Gene amplification
What results from microsatellite instability
Point mutation- based substitutions, microdeletions or insertions
How rare are the events that occur as a result of MIN and CIN
Very rare in normal cells, occuring once every 10 million cell divisions, however the rate in tumour cells may be several thousand times higher.
Where do destabilising mutations come from and how does the cell protect and repair itself
1) DNA damage from exogenous agents - nucleotide excision repair, base excision and double strand break repair.
2) DNA damage from endogenous agents (hydrolysis, oxidation, methylation) - Base excision repair, direct reversal.
3) DNA replication or processing errors - Precursor control, proofreading, mismatch repair
Describe the experiment on cerevisiae yeast investigating RAD9 and temp sensitive cdc9
Cell with RAD9 or rad9 (mutant copy) are healthy in the absence of extrinsic interference (like a lack of cdc9 function - cdc9 encodes DNA ligase essential for completion of DNA
The longer the cdc9ts :rad9 mutant cells are left at the restrictive temp the less viable they become, thus viability in cells experiencing S phase stress require WT rad9
What is a synthetic lethality screen
A loss of viability screen used to screen checkpoint mutants.
What happens when rad9 mutants are exposed to X-ray
They keep dividing and die with fragmented nuclei - this is called mitotic catastrophe
What is associated with premature chomosome condensation
A phenotype associated with a loss of the S/M checkpoint.
What is the action of colchicine and what effect did it have in hamster kidney cells experiencing PCC
It arrests the cells in M-phase, however at the restrictive temperature there is DNA fragmentation and the cells die.
What is the effect of treating cells blocked in S phase with caffeine
Leads to premature chromosome condensation phenotype - caffeine intereferes with a functional pathway in mammalian cells.
How do Xenopus egg extracts will undergo repeated ronds of the cell cycle and how was this shown
Extracts from oocyte and DNA are cultured together. First they are cultured with a32 dATP which is incorporated into DNA during S phase. Extracts of the sample are taken every ten minutes and add the dATP. Does any get incorporated? This gives bursts of DNA replication on the gel
Next done with Cdk1 activity. Add histone H1 to sample extracts to measure its activity, as cells go into mitosis, the amount of Cdk1 activity dramatically increases.
Addition of a DNA replication inhibitor then the kinase activity doesn’t experience the dramatic accumulation seen in the prev extract.
What is the effect of caffeine addition on samples that had DNA replication inhibited
1) The cdk1 cycle and mitosis are restored - dephosphorylation of cdk1 which drives motosis can still occur even when DNA replication is inhibited.
What effect does caffeine have on ATR and what does this mean
Caffeine blocks the activity of the DNA damage sensor ATR (also ATM). This reduces Chk1 activity which would normally act to stim Wee1 and inhibit cdc25 which both act on cdk1.
How does caffeine work
The C terminus of ATR/ATMs have a motif that recognises PI3K
ATR is a protein kinase that is inhibited by caffeine
Chk1 is a mammalian gene which responds to DNA replication stress. When Chk1 becomes phosphorylated it activates Wee 1 and inactivates Cdc25.
These both act on cdk1 to prevent mitosis. Caffeine acts to prevent this inhibition
After detection of ssDNA, what replicative stress mechanisms are initiated by Chk1.
Inhibition of cell cycle progression, stalled fork stabilisation, facilitation of fork restarts, blockage of ORC firing
