8. p53 Flashcards
what can cause DNA damage?
- exposure to mutagens
- oxidative damage
- radiation
what types of genetic lesions can be made?
double strand breaks, single strand breaks, base changes, translocations, amplifications
what do we not want to tolerate and why?
DNA damage as it leads to the accumulation of changes in DNA
and these can lead to cancerous mutations
what enzymes keeps ROS low in cells and how does this differ between people
cytochrome oxidase I
individuals have different alleles of this which make them better or worse at removing ROS
can RO S be tolerated in low levels
yes
what happens if cytochrome oxidase is mutated?
can lead to accumulation of ROS and thus accumulation of DNA damage
>mutations in CO1 are associated with increased risk of cancer
name a group of inherited skin disorders which is characterised by UV sensitivity
Xeroderma Pigmentosum
what happens to the DNA of people suffering from Xeroderma Pigmentosum and what implications does this have?
low levels of UV light causes DNA damage
>they are more susceptible to skin cancer
what genes are associated with Xeroderma Pigmentosum?
there are 7 genes associated with this disease and they are all involved in nucleotide excision repair which is essential for damage made by UV
what is a karyotype test and how might this vary between normal cells and cancer cells?
test to identify and evaluate size, shape and number of chromosomes in cells
>probes specific to chromosomes for hybridisation
>cancer cells are aneuploidy - most chromosomes are present in three copies
> large chromosomal translocations
>within chromosomes - deletions, translocations and point mutations.
when cancer genomes are sequenced and compared to normal genomes, what is hard to say about these mutations?
which mutations are driving the cancer and which are just bystander mutations
what are bystander mutations?
these are mutations which are brought about by random chance and don’t really contribute to the disease
what are drive mutations?
> occur statistically at a greater frequency than would be expected for a standard background mutation
this implies they have been selected for as the disease causing mutations
what does DNA repair ensure?
we don’t accumulate mutations at rates that we should not
what are the four responses a cell could have to DNA damage?
- repair and return to normal
- cell death (cell can be replaced)
- senescence
- mutations - the above responses are all trying to prevent this
how was p53 identified?
identified by doing screens for genes that are changed in cancer
name the disease where p53 was also found to be mutated and is an early onset cancer prone syndrome? how common is this? and why does this happen?
Li-Fraumeni Syndrome
>affects 100 families world wide
>mutation of TS leads to elevated levels of DNA damage - individuals cells are hypersensitive to DNA damage
what does positional cloning do and show and why was this hard in Li-Fraumeni Syndrome
> genomic mapping protocol that identified p53
obtained genomic DNA from affected and unaffected people
look for mutations that are common to affected and not common to in-affected people
indeify candidate gene
not many people as they die young and don’t reproduce
what small DNA viral transforming proteins interact with p53?
HPV E6, SV40 T antigen and Adenovirus E1B
p53 is a tumour suppressor, how is the active species found?
as a homo-tertramer
describe the body plan of p53?
TA domain
DNA binding domains
tetramer formation domain
what happens when p53 is KO in mice? and what does this show?
they are caner prone, significant reduced survival
>the importance of p53 is to maintain a stable genome and prevent cancer.
what is seen when mice are give an extra p5 and exposed to mutagen?
they are even more resistant to cancer
animals like elephants have more cells than us, there cells have been through more divisions but they hardly ever get cancer, why is this?
they have 20 copies of the p53 gene in their genome
give a potential use of genome engineering
give people are additional p53 so that they will be less cancer prone.
what is the frequency of p53 mutation in cervical cancer and why is this?
6%
HPV inactivates p53 and so does not require mutations to lose its function
why are people that have mutated p53 likely to develop cancer?
they can accumulate further mutations which lead to the disease
what type of mutations are normally found in p53? and where are they?
missense mutations, there are point mutations in which a single nucleotide change results in a code that codes for a different amino acid
when mapped back to the 3D structure of the proteins where are these mutation hotspots in p53?
mostly in the DNA binding domain - surface residues that specifically interact with DNA targets of p53, meaning that they can no longer specifically bind and modulate gene expression
what affect can other mutations have on p53, in addition to directly affecting DNA binding site?
can effect how the protein folds and retains its structure. this has implications on how p53 functions and binds target genes.
if you are heterozygous for p53, you would expect to have half WT activity? TRUE or FALSE?
false, you will have less than half WT activity
what implication does p53 being a tetramer have on binding specificity?
the four subunits give DNA binding specificity
when will mutant p53 still be incorporated into tetramer?
when the mutations is in the DNA binding site and so does not affect the overall folding of the proteins and the tetramer forming domain
what happens if a mutant p53 is incorporated into the tetramer?
this will compromise the ability to bind DNA - lowers the affinity of binding
why are p53 mutations in the DNA binding domain dominant negatives?
functionally it dominates the wild type version
when mutations arise in p53 that affect its folding what happens?
are directed to be degraded and so these is loss of p53 activity due to reduced p53
define the consensus sequence of p53?
two degenerate sequences that are separated by 0-2bps - this means that they are found in many places in the genome
bioinformatics identified 524 target sequences of p53, how accurate is this likely to be?
probably no way near as many target genes as are actually affected
>because they way we do bioinformatics excludes quite a lot of the things that are targeted
what are the three classes of genes that p53 binds?
- arrest and repair
- apoptosis
- regulators
what do arrest and repair genes do?
block the cell cycle, allow for repair to take place and then allow the cell cycle to recommence.
give an example of a target p53 genes that causes cells cycle arrest? and what types of arrest does this cause?
p21 - a cdk inhibitor
this causes G1 phase arrest
p21 can block cell cycle, what else can it do?
bind PCNA
what does PCNA stand for?
proliferating cell nuclear antigen
where is PCNA seen?
in the nucleus of proliferating cells
how does PCNA bind DNA?
as a homotrimer
>there is a hole through the middle that accommodates DNA
what is PCNA function?
it acts as a sliding clamp that which functions as a processivity factor of DNA polymerase - its helps DNAP speed along the DNA when replicating
when p21 binds PCNA what does this favour?
p21 binding blocks DNA polymerase binding and processivity
>favouring short repair type DNA synthesis
what does PCNA binding impose?
an S phase block
where can p21 impose cell cycle arrest and what does this depend on?
G1 phase and S phase
this depends on what phase the DNA damage even occurs
name a non-protein target of p53? and what does this do?
miRNA
>this regulates the translation of key cell cycle components
>can down regulate cdk4 and cyclin E translation
>this can impose a G1 block
name the target proteins for p53 that is implicated in G2/M phase arrest? and what do they bind?
14-3-3 proteins
>they bind phospho-serine containing peptides
where is 14-3-3 when not bound it a ligand?
in the nucleus
what happens when the cell cycle is commencing in terms of G2/M phase?
cdc25 is functional and dephosphorylates cdk1
what kinase is activate when there is DNA damage and what does this P to prevent G2/M phase transition?
Chk1
>this P cdc25 at a serine residue
>this becomes a ligand for 14-3-3
>this binding results in 14-3-3 nuclear export of cdc25
>it cannot remove P from cdk1 and so cdk1 remains inhibited
which are the two main transcription targets of p53 in terms of apoptosis? and what are they?
Bcl2 - anti-apoptotic repressed by p53
Bax - pro-apoptotic activated by p53
how is apoptosis triggered?
> Bcl2 stabilises outer mit membrane
cyt c between membranes for oxidative P
increase in Bax/reduction in Bcl2 results in cyt C is released and binds adaptor proteins
this binds inactive pro-caspases
apoptosome is formed and cleaving process commences
how do bcl2 and bax contribute to apoptosis?
> both have TM domains that localise them to outer membrane
Bax multimerising to form pore and let cyt c out
Bcl2 inhibits this pore formation
when there is more bax than bcl2 can inhibit there is greater changes of cyt c release
name another target genes of p53 that regulates cells death, and how does it do it?
PTEN
Akt is a pro-survival, pro-proliferative kinase what can it P to prevent cell death? and what regulates Akt?
pro-caspases to stop them being active
>PTEN inactivates Akt through negative regulation on PIP3
>this allows pro-caspases to be active
what is Bad? and what does it do?
a protein that promotes apoptosis
>it competes of binding of Bcl2 so that it can not for heterodimer with Bax
how can Akt affect bad?
P it and prevent it from interacting with out mit membrane - this prevents apoptosis
name three substrates of Akt?
- pro-caspases
- bad
- MDM2
what is MDM2?
E3 ubiquitin-protein ligase that mediated ubiquitination of p53 and tags it for degradation
by promoting MDM2, what affect does Akt have? and what happen when Akt is inactive?
- promotes degradation of p53
- prevents cell cycle arrest and apoptosis
>when Akt is inactive, MDM2 is inactive
