genome instab + checkpoints Flashcards
what is meant by genome instability?
the elevated risk of genetic alterations
what is the mutator hypothesis?
tumour cells acquire a mutator gene (or lose an anti-mutator gene) which increases the rate of mutation as an early event in cancer development
(explains accumulation of mutations in cancer cells)
Could the absence of P53 change tolerance to mutations?
- may have an effect but must be something before this as there are an accumulation of mutations before TP53 is mutated
what are the 2 forms that tumour cell genome instability takes?
CIN - chromosomal instability
MIN - microsatellite instability
what is meant by chromosomal instability?c
chromosomal aberrations, rearrangements, large chunks of chromosomes deleted/added/moving - macroscopic changes , e.g. LOH, aneuploidy, gene amplificaiton///
what is meant by microsatellite instability?
when specific small sequences change as a result of direct mutation
e.g. point mutation, base substitution, microdeletions/insertions
How common do CIN or MIN occur?
- very rarely in normal cells - maybe once every 10 million divisions
- rate of mutations in tumour cells may be thousands times higher due to the mutator phenotype
where do mutations come from?
- DNA damage by exogenous agents e.g. ionizing radiation, UV, chemicals
- defective repair via NER, BER, DSB (messy via recombination)
- DNA damage by endogenous agents - DNA methylation, oxidation + hydrolysis
- DNA replicaiton issues
- processing errors
- inherited loss of DNA damage response/repair
give some examples of familial disorders caused by genome instability
xeroderma pigmentosum breast cancer ataxia telangiectasia NBS (vunerable to DNA damage)
what is RAD9 a paradigm for?
S/M checkpoint control
what happens when cells lack RAD9 gene (yeast cerevisea) and an extrinsic interference is added?
- there is no difference in the proliferation of normal cells and cells lacking RAD9 - appear to be healthy cells
- If impose an extrinsic interferance e.g. cdc9 temp sens mutant, cells have LOSS OF VIABILITY
what does cdc9 code for?
DNA ligase which stitches fragments of DNA together as they are synthesised
what happens to the FACs when cdc9/rad 9 mutant cells are at a restrictive temperature?
cells should stop at G2, cell cycle arrest as DNA synthsis cannot be completed due to a lack of ligase
- but as there is a loss of RAD9 cells try to undergo mitosis which results in mitotic catastrophy
what can a loss of viability screen show?
can screen for checkpoint mutations
what is a chekpoint control gene?
an extrinsic control gene which ensures viability of the cell is maintained when cells undergo stress
what is meant by synthetic lethality?
2 gene which reduce the viability of cells when both mutated, but do not have this effect when only 1 is mutated
what happned when RAD9 mutated cells are exposed to stress/x-rays?
Despite DNA damage, the cells keep dividing and die with fragmented nuclei - still undergo mitosis
- results in MITOTIC CATASTROPHY
what does PCC stand for?
premature chromosomal condensation
what is PCC associated with ?
a phenotype associated with loss of the S/M checkpoint
- chromosomal instability occurs prematurely, cells are going into mitosis before they have finished DNA replication - results in mitotic catastrophy (cells die)
(observed in hamster ovary cells)
what happens when there is a DNA replication block and caffeine is added?
When caffiene is added cells still undergo mitosis prematruely - get PCC and fragmented dna - mitotic catastrophy
- uncouples mitosis from completetion of DNA replication - as mitosis occurs even though DNA replication is not completed
what was the target of caffeine found to be?
- knew it was to do with protein synthesis, as when protein synthesis was blocked by inhibitors, the effect f caffeine was blocked
- ATR?
how does coupling of mitosis to the completion of DNA replication occur?
-occurs via the modulation of a tyrosine kinase which phosphorylates P34cdc2 (CDK1)
what happens to cdk1 when caffeine is added during an interphase block?
- when caffiene is added cdk1 becomes dephosphorylated (lose inhibitory phosphates)
- cdk1 becomes activated and induces mitosis (premature)
- mitotic catastrophy
what effect does ocacdaic acid have?
can have the same effect of caffeine
- induce premature mitosis - mitotic catatrophy
what is ATR?
A caffeine sensitive DNA activation protien kinase with substarte specificity
- inhibited when caffiene is added
- ATM/ATR same thing
- phosphorylates chk1
what happens when chk1 is phosphorylated by ATR during DNA damage?
Chk1 upregulates Wee1K
Chk1 dwonregulates cdc25
- this results in the inhibition of cdk1 - so mitosis cannot occur
how can replication stressed be sensed?
by the prescence of ssDNA
- can be sensed by RPA - when a lot of RPA can recruit proteins like ATR - phosphoryltes chk1, cell cycle arrest - mitosis cannot occur
what does phosphorlated chk1 do?
- inhibit cell cycle progression
- stabilise staled forks
- facilitate fork restart when ready
- block origin firing
