L10 - Genomic Instability Flashcards
Describe the disovery of p53
In 1975 - thought to be the basis of the decision to replicate the DNA
What virus was used to help discover p53
Papovirus - SV40
Describe the normal distribution of SV40 protein
Reside in the nucleus
Advantage of SV40 proteins
Immunogenic - they are easy to raise antibodies against
What is the relevant viral protein from SV40
Large T
Describe what happens when you transfect cells with viral SV40
What is the experimental method required to see this
Can see that proteins are constantly undergoing protein synthesis
Shown through the addition of a radio-AA
What was the next direction after the SV40 transfection
To ask wehther large T interacted wth any other host proteins
Results of immnoprecipitation experiemnts of cells TF with SV40 large T
In presence of antiserum or antiserum from control animal pre-immunisation
Only immunopreciptate in transformed SV40 cells where antibody present
Band around 54kD = p53
Purpose of a cellular transformation assay
Allows you to determine if cell is transformed in the presence of an oncogene
How would you tell if a cell had been transformed
If the cell shows anchorage independent growth … transformation … clone into an immune compromised mouse
Ras + P53 mutant
Normal number of colonies
CONTROL
Ras+P53
P53 FROM TISSUE CULTURE CELLS
Increased numbr of colonies … looks like p53 acting as an oncogene
BUT
Tissue culture cancer cell line - vast majority of p53 has been lost due to genomic instability
(In the case from the paper there was a Val135 mutation)
Ras + WT (true) P53
No colonies
Onogenic function of Ras has been supressed
P53 BEHAVES AS A TUMOUR SUPRESSOR GENE
What cancers display a p53 mut
ALL
What happens usually when TSGs are deleted in the embryo
What is done to get around this?
Is this the case for p53
Embryonic lethality
- Since many TSGs are regulators of cell numbers
Conditional KO approaches
Not the case for P53
Why are the Kaplan Meier plots for p53 mutations unusal?
If P53 is a TSG would expect to see Knudsens rules followed… but they aren’t
What is an epititope map
Pannel of antibodies that recognise p53 but each recognises it at a different site (epitope)
Describe the experiments that lead t the conclusion of p53 having a short half life
Label cells with radio-methionine
Harvest cells at 1 hour
Blot for p53 with Pab421
Can see after around 45 minutes alsmost all 421 expression is lost
p53 HAS A HIGHTURNOVER RATE
Descibe the effect of LargeT/p53 interaction has on Pab426 binding
With large T present p53 cant bind anymore
Describe the experiments looking at how much p53 was in the cells
Control vs large T TF
In normal 421 detects p53
But in SV40 transformed cells - 246 detectable p53 in non transfect but in SV40 246 cant detect p53
Is it the levels of p53 that changes in the SV40 transformed cells?
No…
Using a-p53 for a different epitiope … shows there is equal ammounts of p53
What sort of mutations occurs in p53
What is unusual about this?
Most are missense mutations
There is an unexpected bias in the mutations
Domains within p53 affect transcriptional activation of ….
Gal4
Describe the experiments looking to see if p53 engaages with DNA as a transcritpion factor
STRUGGLES TO DEMONSTRATE DNA BINDING
If have DNA binding domain and transcriptional activating domain = lots of gene expression
Take VP16 and fuse p53 to gal4 construct
If just use N-terminal p53 can show transcriptional activation
Suggests part had transcriptional activation
Evidence that p53 acts as a tetramer
Migrates as a 45kd and 145kd protein during glucose sucrose density gradient centrifugation
What does the tetrameric strucuture explain about p53 acting unusally as a TSG
In hets
Unlikely to get 4 subunits of total WT (only 1/16 chance)
Likely to have AT LEAST one mutant subunit (15/16)
Wt P53 is tthe
Guardian of the genome
mutant p53 at Pab246 site…
Loses its guardian role
When is the guardian role of p53 lost?
Lost in the presence of large T or through missense mutations
What happens to p53 levels if you irradiate the skin withUV
How was this seen
Increase in the levels of p53
Western blot experiments
Describe an experiment to see what genes are promoted by p53
Null cell line is easy to come by since p53 is commonly mutated
Insert p53 into null line under control of glucocorticoid promoter
Apply dexamethasone …
Look for differences in the transcriptional output
ISOLATE mRNA
Subtractive hybridisation
Essentially to identify which genes arent present
What was a gene that p53 promotes
WAF1
WAF1 aka
p21
What is p21
Effects of p21
CDK inhibitor
Inhibits CDK4/2/1
Controls the G1 –> S phase progression through p53
Types of genes that p53 upregulates
Growth arres genes
DNA repair genes
Regulators of apoptosis
Anti-angiogenic proteins
Specific alarm signals provoke p53 mediated
Inhibition of proliferation
Triggering of apoptosis
Cytostatic and pro-apoptotic functions of p53 represent a threat to incipient cancer cells for which the following are necessary conditions
Hypoxia
Genomic damage
Signalling excess
Inactive forms of p53 enable
Oncogene activation wo apoptosis
Higher tolerance of anoxia
Sloppy oversight of chromosomee inegrity
What is the double minutee phenotype
Double minute chromosomes seen in fibroblasts NIH-3T3
Cells able to spontaenously transform and produce tumours
Identified and cloned genes OE in these cells
What genes are OE in the double minute phenotype
Mdm1-3
OE Mdm1-3 sufficient to cause
Transformation
Coprecipitation of ___ and ____
Mdm2 and p53
Mdm2 plays an important role in …
Inducing the destruction of p53
Increasing mdm2 lead to …… in a …
P53 not being present
CONCENTRATION DEPENDENT MANNER
What was the control when looking at the p53 / mdm2 relationship
Using mutant p53 which is lacking the N-terminal
Mdm2 is a
Ubiquitin ligase
What does Mdm2 do to p53
polyubiquitinate marking for proteasomal degradation
if you increase Mdm2 … p53 ______
What is Mdm2 acting as`
p53 will decrease
Mdm2 acting here as an oncogene
Give some examples of dependency points … explain each one
Are dependency events absolute?
Bacteriophage assembly
Later component assembly dependent on the correct assembly of earlier ones
Drosophila polytene chr
Huge - exist since multiple reps without division , SHOWS depdency events are not aboslute
Polyploidy
Multipel chr per cell
SHOWS depdency events are not aboslute
NO
Outline the paradigm for S/M checkpoint control in Yeast
Cells lacking in Rad9 healthy in the absence of extrinsic interference
What is CDC9
What is the effect of losing Cdc9?
DNA ligase - responsible for sealing the nick between okazaki fragments
WO Cdc9 cant get a sigar phosphate backbone all of the way through the molecule
Describe the mutation in the Yeast paradigm
What were the mutants crossed with
Temperature dependent Cdc9 mutation
Too high - null
Permissive - Cdc9 will work
Done this was since conventional Cdc9 KO would be lethal
Then cross Cdc9 mutants with Rad9 mutants
Descirbe the Cdc9 ts mut/ Rad9
What would you expect to seen … what is actually seen
At the permissive temperature
At the restrictive temperature
Normal
Would expect them to be stuck in S phase
Since checkpoint contols should prevent the advance of the cell cycle when replication is incomplete
BUT ACTUALLY SEE CELLS ADVANICING INTO M PHASE
Describe the viability determination of these cells
Wt
Cdc9 ts - stuck in S phase but still viable
Rad9 - viable
Cdc9ts Rad9 - because no Rad9 cells going into G2 and M without properly replicated DNA
Rad9 is the ….
Founder of the S/M checkpoint in yeast
Describe the experiments to demonstrate the S/M checkpoint in mammalian cells
Hamster cells with ts mut
At restricitve temp ith DNA rep inhibitor of colchicine (blocks cells in M)
Restrive - fragmentation of chromosomes caused by improper segregation
Permissive - condensed chromosomes
Loss of checkpoint control may also be induced with
Caffenine
All metozoan systems have a ______- componenet at the S/M checkpoint
Caffeine sensitive
What does caffeine interfere with
Transducers and effectors
Describe the replication fork sensors
At the replication fork …
Stalling due to lesion leads to the assembly of a complex sensor system
Important to ensure integrity during replication
Occurs ALOT
ATR is ________ sensnitve and is related to ____
Caffeine sensitive
Rad3
ATR is a
Protein kinase - very complex strucutre and is an atypical protein kinase
TRANSDUCTION DEVICE WHICH IS CAFFEINE SENSITIVE
Describe the mechanism involving ATr that occurs following DNA damage
Replication stress leads to ATR being turned ON
Assembly of the sensor
Turns on ATR
Turns on Chk1
+wee1 , -cdc25
Cdk1 is OFF and entry into mitosis is blocked
What does the inherited loss of checkpoint proteins demonstrate
The role of checkpoints in supressing cancers (spontaneous and inherited)
Checkpoint signalling is essential for
P53 function
What is cisplatin
Chemotoxic agent
Is a chemotherapy drugs used to solid tumours - induces apoptosis
Describe what happens when cisplatin is applied
p53 is phosphorylated at serine-15 - N terminal P53 (criticial for Mdm2 interaction) - this prevents the binding of Mdm2 and p53 is not degraded
Describe what happens when a dominant negative form of ATR is used
No phos and activation of p53
Mdm2 able to ubiquitanted and degrade p53
