8. p53

Question 1

what can cause DNA damage?

Answer 1

• exposure to mutagens
• oxidative damage
• radiation

Question 2

what types of genetic lesions can be made?

Answer 2

double strand breaks, single strand breaks, base changes, translocations, amplifications

Question 3

what do we not want to tolerate and why?

Answer 3

DNA damage as it leads to the accumulation of changes in DNA
and these can lead to cancerous mutations

Question 4

what enzymes keeps ROS low in cells and how does this differ between people

Answer 4

cytochrome oxidase I

individuals have different alleles of this which make them better or worse at removing ROS

Question 5

can RO S be tolerated in low levels

Answer 5

yes

Question 6

what happens if cytochrome oxidase is mutated?

Answer 6

can lead to accumulation of ROS and thus accumulation of DNA damage
>mutations in CO1 are associated with increased risk of cancer

Question 7

name a group of inherited skin disorders which is characterised by UV sensitivity

Answer 7

Xeroderma Pigmentosum

Question 8

what happens to the DNA of people suffering from Xeroderma Pigmentosum and what implications does this have?

Answer 8

low levels of UV light causes DNA damage

>they are more susceptible to skin cancer

Question 9

what genes are associated with Xeroderma Pigmentosum?

Answer 9

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

Question 10

what is a karyotype test and how might this vary between normal cells and cancer cells?

Answer 10

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.

Question 11

when cancer genomes are sequenced and compared to normal genomes, what is hard to say about these mutations?

Answer 11

which mutations are driving the cancer and which are just bystander mutations

Question 12

what are bystander mutations?

Answer 12

these are mutations which are brought about by random chance and don’t really contribute to the disease

Question 13

what are drive mutations?

Answer 13

> 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

Question 14

what does DNA repair ensure?

Answer 14

we don’t accumulate mutations at rates that we should not

Question 15

what are the four responses a cell could have to DNA damage?

Answer 15

• repair and return to normal
• cell death (cell can be replaced)
• senescence
• mutations - the above responses are all trying to prevent this

Question 16

how was p53 identified?

Answer 16

identified by doing screens for genes that are changed in cancer

Question 17

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?

Answer 17

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

Question 18

what does positional cloning do and show and why was this hard in Li-Fraumeni Syndrome

Answer 18

> 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

Question 19

what small DNA viral transforming proteins interact with p53?

Answer 19

HPV E6, SV40 T antigen and Adenovirus E1B

Question 20

p53 is a tumour suppressor, how is the active species found?

Answer 20

as a homo-tertramer

Question 21

describe the body plan of p53?

Answer 21

TA domain
DNA binding domains
tetramer formation domain

Question 22

what happens when p53 is KO in mice? and what does this show?

Answer 22

they are caner prone, significant reduced survival

>the importance of p53 is to maintain a stable genome and prevent cancer.

Question 23

what is seen when mice are give an extra p5 and exposed to mutagen?

Answer 23

they are even more resistant to cancer

Question 24

animals like elephants have more cells than us, there cells have been through more divisions but they hardly ever get cancer, why is this?

Answer 24

they have 20 copies of the p53 gene in their genome

Question 25

give a potential use of genome engineering

Answer 25

give people are additional p53 so that they will be less cancer prone.

Question 26

what is the frequency of p53 mutation in cervical cancer and why is this?

Answer 26

6% | HPV inactivates p53 and so does not require mutations to lose its function

Question 27

why are people that have mutated p53 likely to develop cancer?

Answer 27

they can accumulate further mutations which lead to the disease

Question 28

what type of mutations are normally found in p53? and where are they?

Answer 28

missense mutations, there are point mutations in which a single nucleotide change results in a code that codes for a different amino acid

Question 29

when mapped back to the 3D structure of the proteins where are these mutation hotspots in p53?

Answer 29

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

Question 30

what affect can other mutations have on p53, in addition to directly affecting DNA binding site?

Answer 30

can effect how the protein folds and retains its structure. this has implications on how p53 functions and binds target genes.

Question 31

if you are heterozygous for p53, you would expect to have half WT activity? TRUE or FALSE?

Answer 31

false, you will have less than half WT activity

Question 32

what implication does p53 being a tetramer have on binding specificity?

Answer 32

the four subunits give DNA binding specificity

Question 33

when will mutant p53 still be incorporated into tetramer?

Answer 33

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

Question 34

what happens if a mutant p53 is incorporated into the tetramer?

Answer 34

this will compromise the ability to bind DNA - lowers the affinity of binding

Question 35

why are p53 mutations in the DNA binding domain dominant negatives?

Answer 35

functionally it dominates the wild type version

Question 36

when mutations arise in p53 that affect its folding what happens?

Answer 36

are directed to be degraded and so these is loss of p53 activity due to reduced p53

Question 37

define the consensus sequence of p53?

Answer 37

two degenerate sequences that are separated by 0-2bps - this means that they are found in many places in the genome

Question 38

bioinformatics identified 524 target sequences of p53, how accurate is this likely to be?

Answer 38

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

Question 39

what are the three classes of genes that p53 binds?

Answer 39

* arrest and repair * apoptosis * regulators

Question 40

what do arrest and repair genes do?

Answer 40

block the cell cycle, allow for repair to take place and then allow the cell cycle to recommence.

Question 41

give an example of a target p53 genes that causes cells cycle arrest? and what types of arrest does this cause?

Answer 41

p21 - a cdk inhibitor | this causes G1 phase arrest

Question 42

p21 can block cell cycle, what else can it do?

Answer 42

bind PCNA

Question 43

what does PCNA stand for?

Answer 43

proliferating cell nuclear antigen

Question 44

where is PCNA seen?

Answer 44

in the nucleus of proliferating cells

Question 45

how does PCNA bind DNA?

Answer 45

as a homotrimer | >there is a hole through the middle that accommodates DNA

Question 46

what is PCNA function?

Answer 46

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

Question 47

when p21 binds PCNA what does this favour?

Answer 47

p21 binding blocks DNA polymerase binding and processivity | >favouring short repair type DNA synthesis

Question 48

what does PCNA binding impose?

Answer 48

an S phase block

Question 49

where can p21 impose cell cycle arrest and what does this depend on?

Answer 49

G1 phase and S phase | this depends on what phase the DNA damage even occurs

Question 50

name a non-protein target of p53? and what does this do?

Answer 50

miRNA >this regulates the translation of key cell cycle components >can down regulate cdk4 and cyclin E translation >this can impose a G1 block

Question 51

name the target proteins for p53 that is implicated in G2/M phase arrest? and what do they bind?

Answer 51

14-3-3 proteins | >they bind phospho-serine containing peptides

Question 52

where is 14-3-3 when not bound it a ligand?

Answer 52

in the nucleus

Question 53

what happens when the cell cycle is commencing in terms of G2/M phase?

Answer 53

cdc25 is functional and dephosphorylates cdk1

Question 54

what kinase is activate when there is DNA damage and what does this P to prevent G2/M phase transition?

Answer 54

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

Question 55

which are the two main transcription targets of p53 in terms of apoptosis? and what are they?

Answer 55

Bcl2 - anti-apoptotic repressed by p53 | Bax - pro-apoptotic activated by p53

Question 56

how is apoptosis triggered?

Answer 56

>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

Question 57

how do bcl2 and bax contribute to apoptosis?

Answer 57

>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

Question 58

name another target genes of p53 that regulates cells death, and how does it do it?

Answer 58

PTEN

Question 59

Akt is a pro-survival, pro-proliferative kinase what can it P to prevent cell death? and what regulates Akt?

Answer 59

pro-caspases to stop them being active >PTEN inactivates Akt through negative regulation on PIP3 >this allows pro-caspases to be active

Question 60

what is Bad? and what does it do?

Answer 60

a protein that promotes apoptosis | >it competes of binding of Bcl2 so that it can not for heterodimer with Bax

Question 61

how can Akt affect bad?

Answer 61

P it and prevent it from interacting with out mit membrane - this prevents apoptosis

Question 62

name three substrates of Akt?

Answer 62

- pro-caspases - bad - MDM2

Question 63

what is MDM2?

Answer 63

E3 ubiquitin-protein ligase that mediated ubiquitination of p53 and tags it for degradation

Question 64

by promoting MDM2, what affect does Akt have? and what happen when Akt is inactive?

Answer 64

- promotes degradation of p53 - prevents cell cycle arrest and apoptosis >when Akt is inactive, MDM2 is inactive