The cell cycle

Question 1

what are the 4 stages if the cell cycle and what happens in them?

Answer 1

G1: organelles and other cellular parts will duplicate but not the chromosomes
S phase: the chromosomes are duplicated
G2: cell checks for errors during duplication
Mitosis: segregation cell divides

Question 2

what are the 4 key things each cell cycle transition must be?

Answer 2

• unidirectional
• robust
• sequentially linked
• linked to cellular events

Question 3

which transition means that the cell has committed to a new cell division?

Answer 3

G1/S

Question 4

what are cellular events that must be ensured in order for the G1/S phase to happen

Answer 4

• the cell has grown enough
• the cell has enough nutrients
• the cell has enough mitogens

Question 5

what must have happened in order for the G2/M transition to happen?

Answer 5

the cell must have completed DNA replication and resolved DNA damage

Question 6

what does having robust activation mean?

Answer 6

• an all or nothing signal- no midway and is not reversible

Question 7

what is key to ensuring a robust signal in cell cycle transitions?

Answer 7

• the integration of positive feedback loops. For example, once a signal pathway is activated, If a component can activate the expression of itself and also the component before it, there will be a huge increase in the pathway downstream, meaning essentially an all or nothing signal once activated.

Question 8

How can a cell cycle transition signal ensure it is sequentially linked?

Answer 8

• as a function of its activation it must essentially inactivate itself. This will ensure that the transition will not happen again until the previous transition has occurred. Often the last component of the pathway after the positive feedback loop inhibits its activator.
• this occurs by integrating negative feedback loops that have a lag on the activation of the signal
• its activation must also depend on components of the previous phase.

Question 9

what is the key enzyme in cell cycle transitions?

Answer 9

cyclin-dependent kinase

Question 10

how does CDK act?

Answer 10

it binds to cyclin and then it can phosphorylate downstream proteins which it acts to activate or inactivate

• in different phases it binds different cyclins
• the relative levels of cyclins then regulate CDK activity

Question 11

in s phase in human, what type of cyclins are expressed?

Answer 11

cyclin A

Question 12

in M phase in humans, what type of cyclins are expressed?

Answer 12

cyclin B

Question 13

in G1 in humans what type of cyclins are expressed?

Answer 13

cyclin D

Question 14

in G1/S phase in humans, what cyclins are expressed?

Answer 14

cyclin E

Question 15

what are the dynamics of CDK within the cell cycle?

Answer 15

it regulates the level of cyclins and it is in turn regulated by levels of cyclins

Question 16

what is cell cycle regulate transcription?

Answer 16

it is essentially the idea within different phases, there are contrasting transcription profiles, with around 1000 genes being upregulated or down regulated in each phase. These genes are then down regulated with the onset of the next stage

Question 17

how does the concentration od CDK change throughout the cell cycle?

Answer 17

remains the same - cell cycle transitions depend on cycle levels

Question 18

how are all other cyclins other than G1 cyclins kept down regulated during G1? (3)

Answer 18

• expression of the major cyclin genes is suppressed by the inhibitory gene regulatory protein
• APC specifically targets S and M cyclins (but not G1/S cyclins) for degradation
• there are high concentrations of CDK inhibitors in G1

Question 19

how is G1/S expression initially allowed to start within G1?

Answer 19

• due to APC not targetting G1/S proteins for degradatio, when signals from outside the cell such as mitogens trigger the upregulation of G1/S cyclins, they are able to increase. This means the G1/S can act to trigger the destruction of cdikinhibitor proteins and inactivate the APC complex

Question 20

what is the main role of G1/S gene targets?

Answer 20

• to trigger the destruction of CDK inhibitor proteins
• inactivate the APC complex
• both these things allow S cyclins (cyclin E) to increase

Question 21

what is the general role of S phase cyclins? what happens to G1/S cyclins as it proceeds?

Answer 21

to phosphorylate proteins that initiate chromosome duplication.
G1/S cyclins then begin to trigger their own demise via G1/S-cdk action

Question 22

what do M phase cyclins importantly activate? how does this end the cycle?

Answer 22

the activation of the APC complex which trigger metaphase to anaphase transition by stimulating the degradation of the proteins that hold the sister chromatin together

• APC also instigates the destruction of S and M cyclin genes expression and increases production of cdi inhibitor in late mitosis
• the resulting inactivation of sdks allows dephospho rylation of their mitotic targets, which is required for spindle disassembly and completion of M phase.

Question 23

how is cdk activity in yeast different to multicellular eukaryotes?

Answer 23

• in budding yeast and fission yeast, all cell cycle events are controlled by a single essential cdi called cdk1
• in multicellular eukaryotes cdk1 and cdk2, cdk1 acts primarily in M phase and cdk2 in s phase.
• animal cells also contain two sdks (4 and 6) that are important in regulationg entry into the cell cycle in response to extracellular factors

Question 24

how is cdk activated?

Answer 24

in order to be activated it requires a conformational change in its active site: this happens when cyclins bind

Question 25

what is the budding yeast equivalent of vertebrate G1/S cyclins (cyclin E)?

Answer 25

Clin 1 and Clin2

Question 26

other than targeting the upregulation of S cyclins, what else do G1/S cyclins do?

Answer 26

• the duplication of the centrosome

Question 27

what is the general role of Mcyclin-cdk complexes?

Answer 27

• the stimulate the assembly of mitotic spindle and the alignment of sister chromatid pairs on the spindle at metaphase. their destruction in anaphase bring on mitotic exit and cytokinesis

Question 28

other than binding cyclin, how is cdi activated and is this reversible? what enzyme does this?

Answer 28

• it needs to be phosphorylated but this isn’t reversible so does not change throughout the cycle. CDK activating enzyme does this.

Question 29

describe how a robust G2/M transition is orchestrated via regulation of M cyclin-CDK

Answer 29

• M phase cyclins begin to be upregulated in S phase. CDK1 has a high binding affinity for cyclin B in vert.
• if cyclin B was not inhibited during S phase, there would be a linear increase in activity which would not be robust
• thus a mechanism must be introduced to confer a bistable switch.
  this is achieved by Wee1 and cdc25. Wee1 inhibits CDK1 via phoshprylation and cdc25 is a phosphatase that removes this inhibition.
• CDK1-cyclin B inhibits wee 1 and activates cdc25
• initially, when M cyclins (cyclin B) is absent, Wee1 activity is very high and cdc25 activity is low.
  as cyclin-B cdi levels increase, a large amount builds up
• instead of a threshold level being reached, it is thought that multiple regulatory elements work to instigate the bistable switch but it is not well understood
• one idea is that cyclin-A-cdk2 which is active in G2 can phos and partly activate cdc25. This activates cyclin-b-cdk1 which then activates more cdc25 and inhibit wee1- and a feedback machoism works

Question 30

explain how cyclins can be specialised to particular functions

Answer 30

• in yeast it is thought that S and M cyclins may act in a concentration dependent manner, activating different targets at different levels- there is a lot of functional overlap of these in yeast
• cyclins can interact directly with the substrates of associated cdk (cyclin A and not cyclin B interact with p107, a transcriptional regulator)
• they can direct CDK complex to specific sub cellular locations (cyclin B1 targets the nuclear lamina and phosphorylates the protein within it to trigger its breakdown)
• binding of adaptor proteins that promote phos of targets

Question 31

what is the name of the cdk inhibitor that acts during G1 in budding yeast? how does it act within G1? how do they come to be inhibited in late G1?

Answer 31

Sic1- they act to inhibit major S and M CDK complexes- does not inhibit G1/S cyclins. as G1/S levels rise in response to external factors, They bind with CDK and phosphorylate Sic1, which targets it for degradation

Question 32

how does the CDK inhibitor in G1 come to be degraded? what is it called?

Answer 32

in budding yeast- Sic1- doesn’t inhibit G1/S cyclin CDK but does S and M. As G1/S increase n response to external factors, They bind with CDK and phosphorylate Sic1, which targets it for degradation

Question 33

has a mammal homologue of sic1 been found? is there anything similar?

Answer 33

no in mammals there is p27 which governs CDK activity in G1 but it inhibits G1/S thus must be removed in late G1- this is achieved by G1-CDKs removing p27 from G1/S CDKs, which it is also destroyed by (?)

Question 34

when is proteolysis critical?

Answer 34

metaphase to anaphase

Question 35

why is the destruction of cyclins important in metaphase to anaphase?

Answer 35

• mitotic exit crucially depends on destruction of mitotic cyclins and proteins that control sister chromatic cohesion
• it helps to establish a low CDK activity in G1

Question 36

what are the two protein ligases critical within the cell cycle transitions and when do they work?

Answer 36

• SCF in the G/S transition

- APC in metaphase to anaphase

Question 37

what is the role of SCF?

Answer 37

• at the G1/S transition it degrades CDK inhibits and G1/S cyclins to ensure the S phase transition is stable
• SCF is activates by phosphorylation by CDKs - thus G1/S trigger their own demise

Question 38

what re the two main targets of the APC complex and why are they significant?

Answer 38

• degrades securin, which allows separase to act which destroys sister chromatid cohesion
• degrades S and M cyclins, degradation inactivates S and M cdks, allow completion of mitosis

Question 39

how is APC activated?

Answer 39

cdc20 or cdh1 binding

Question 40

when do they two different binding partner binds to APC?

Answer 40

• cdc20 binds in metaphase to anaphase transition

- cdh1 activates APC I late mitotic and early G1 to maintain cyclin destruction until entry into the cell cycle

Question 41

what is the task of APC cdc20 during M phase and how is it activated?

Answer 41

• M-cdks activates APC by phos which enhance cdc20 binding. APCcdc20 targets securin and M cyclins to degrade, thereby inactivating M-cyclins. APC activation and M-cyclin destruction occurs after a delay.
• because APC cdc20 depends on m-cyclin and it degrades m-cyclin, it triggers its own demise and cdc20 eventually does not bind anymore

Question 42

what allows cdh1 to bind to the APC complex in early G1 and not in S or M phase?

Answer 42

• CDKs phos cdh1 stopping it from binding APC
• after APCcdc20, there is no cdk activity
• cdh1 become unphos and so can bind during G1
• cdh1-APC destroy S and M cyclins but not G1/S (like sic1) and so these can rise.
• as they rise they phosphorylate cdh1 and stop it binding to APC, thus preventing its function

Question 43

describe how key gene regulatory proteins become expressed in the G1/S transiiton in yeast and how this ensures one direction of the cycle

Answer 43

before start, these factors are inhibited by Whi5. in the late G1, the activity of G1 cdk promotes the inhibitory phosphorylation of Whi5, thereby unleashing SBF and MBF. this results in an increase in G1/S gene expression, which includes genes encoding G1/S and S cyclins. (g1/S cyclins then activate more cdi-cyclin G1 activity and SBF to promote a psoitve feedback loop. Thus the activation of SBF and MBF promotes G1/S and S phase cdk activities and at the same time provides some of he enzymes and raw materials needed ot begin S phase.
- Sic1 becomes inhibited as a result, this also allows S and M cyclins to inhibit SBF- turning off the switch

Question 44

what is the equivalent of SBF and MBF in metazoans?

Answer 44

E2F family and pRb is Whi5

Question 45

what is the long standing idea of cell checkpoints and why is this wrong ?

Answer 45

• the idea was that for cancer to develop, all the check points should be compromised - but cancers rely on the DNA damage checkpoint which it needs to prevent replication stress which would otherwise cause catastrophe and cell death

Question 46

what transition is implicated in every type cancer?

Answer 46

an upregulation of G1/S genes, if they are upregulated, as soon as you finish on mitosis you commit to the next cycle- proliferation. particularly E2F family which is required for the the activated of G1/S transcript (SBF and MBF homologue).

Question 47

how is the E2F pathway implicated in cancers?

Answer 47

it regulates G1/S gene expression, mutations have been found in every step of the pathway in cancers. These mutations results in an increase of E2F activity

Question 48

what does increased proliferation cause?

Answer 48

• increase replication stress because there is unschedules S phase entry and the cells are not prepared to start replication their DNA,. This leads to replication forks stalling which leads to exposure of single stranded DNA which is fragile and becomes damaged.

Question 49

at what checkpoint will DNA damage be detected?

Answer 49

DNA replication stress checkpoint

Question 50

what is the DNA replication stress pathway?

Answer 50

ATR activates CHK1 which blocks M phase entry and acts to prevent DNA damage

Question 51

why do cancer cells rely on DNA replication stress checkpoint?

Answer 51

the DNA replication stress response cannot be comprimised in can not be compromised. The cancer cells rely on this check point. Because if they do ot have this check point, and the replication stress is not softened, there will be incredible genomic instability and the cell will simply die.

Question 52

what is the role of DNA replication stress check point?

Answer 52

Part of this response is to regulated cell cycle progression, also to sabilise replication fork and restart the replication once replication stress resolved. It also inhibits new replicaition origin is initiated.

Question 53

what was initially found in cells that were undergoing replication stress which lead them to E2F?

Answer 53

they found from their yeast work that DNA replication check point maintains G1/S transcription when activated. They found that when you have dna replication stress, the G1/S transcription factors are maintained rather than downregulated (find this paper and look at their figure). They then wanted to check if this was the case in human cells. They took human cells and arrested them in G1. Then they synchronously released them into the cell cycle. then they looked at a specific G1/S tragte, cyclin E, and they found that untreated cells normally have cyclin E activated, peaks at around 16 hours, and then is unactivated. They did the same with and treated cells with hydroxyurea which results in dNTP depletion which causes replication stress. and they found that cyclin E was increased and maintained when it should have been downregulated.

Question 54

when they found that replication stress cells were upregulated G1/S proteins, how did they show this was in a checkpoint dependent manner?

Answer 54

they asked if this was dependen ton the checkpoint protein kinases, specificlaly Chk1. They then treated cells with an inhibitor of Chk1, and they found that even thought the cells are treated and experiencing replicaton stress, they transcriptionaly response of cyclin E is not initiated. This showsthat in human cells that G1/S transcription in the cases of replication stress, is maintained in a checkpoint dependent manner.

Question 55

how did the discover that replication stress-induced upregulation of G1/S proteins was caused by E2F?

Answer 55

in yeast they showed that the way this G1/S transition is maintained if via the inactivation of the negative feedback loops that normally downregulate G1/S transcription by the checkpoint protein kinases.
they wanted to see if this was right in mammalian cells.
the negative feedback loop in mammalian cells relies on E2F6. It is a repressor that is a G1/S traget itself. WHen you activated G1/S transcription you start to accumukate E2F6, which binds and represses transcription activity of E2F1 SO they wanted to see if the checkpoint could inhibit E2F6. They looked at a whole set of E2F targets and looked at them in treated and untreated cells. They found that all these targets were induced in the case of rpelication stress (treated). they then checked these cells were regulated by E2F6 by applying siE2F6 to the cells and predicted that if these things were normally being repressed by E2F6, then when E2F6 is inhibited, they should be upregulated, and they were.
overall, this suggested that in response to replication stress, E2F6 is inactivated to allow the upregulation of G1/S protein levels

Question 56

how did they test what the contribution of maintain E2F1 dependent transcription when there is replication stress? what did they find?

Answer 56

• they CA inhibited E2F1 when there was replication stress
• they developed a cell line in which they could over express E2F6, if you over express E2F6, chk1 cant inactivate E2F6 and therefore it cannot maintain E2F dependent cell cycle progression but it can still initiate the checkpoint response. This allowed them to ask the question, what is the contribution of maintaining E2F dependent transcirption to the checkpoint response.
  The first thing they tested was viability: they used the same system: in untreated cells, if you overexpress ETF6 they are still viable. But i fyou do this in the presence of replication stress: Doxy, you see that see that a lot less cells survive.
  This shows that the upreglation of E2F1 in response ot rpelication stress is important to cell survivial.

Question 57

how did they prove that the upregulation of E2F was required to prevent DNA damage during replication stress?

Answer 57

They then used single cell immuno live microscopy, they looked at single cells that are stained with a marker for replication stress, but also with a marker for DNA damage. They treated cells for 2 hours with replication stress, they saw that some of these cells that suffer from replication stress encounter DNA damage. These cells have an intact DNA damage checkpoint response. They then did the same experiment but over expressed E2F6 so yo uinhibit E2F1 activity, you see that the majroity of cells that encounter replication stress, have DNA damage. This shows that E2F1 actvity is required to prevent DNA damage and thus for cell survivial.

Question 58

what is and isn’t the specific function of E2F increase in response to replication stress check point?

Answer 58

its not involved in regulating cell cycle progression
or in in replication origin formation
it is essential for rep form stability and restart after repair of replication forks

Question 59

how did they shows that E2F upregul in response to replication stress checkpoint activation was required for replication folk formation and restart?

Answer 59

• one way to look at replication fork stability and restart is to look at DNA fiber analysis. This type of analysis depends on modified nucleotides. you feed cells a modified nucleotide that they will incorporate into newly synthesized DNA, then you have a specific antibody that will recognise this DNA. Then after some time you can switch to another modified nucleotide and this will be incorporated.
  then you spread out your DNA and find out when your different nucleotides were incorporated. you can use this to find fork speed and forms of ongoing replication
• hey exposed cells to one modified nucleotide, then you wash this away, then you treat cells with hydroxyurea to induced replication stress for 2 hours. Then you wash this out and put int he second nucleotides. This allows you to look at replication dynamics before and after replication stress.
  they do this control conditions with an intact checkpoint (E2F1 activity intact), and in the presenceo fthe siE2F6 which is the checkpoint inhibitor- so there is no replication stress response while there is replication stress).
  if you see both colours it means that replication can be initated after replication stress. if you see only one colour then there is no replication. They also looked at the fibre length before or after rpelication stress with and without a checkpoint. They found that the fibre length is significantly shortened if you dont have a check point.
• they then tested whether if they dont have a checkpoint, but maintain E2F dependent transcription, is the cell capable of reinitaing replication. They do this by treating cells with UCN01 so that they have no checkpoint response, and with siE2F6 so that they can maintain E2F1-dependent transcription. And this shows that even if they odnt have a checkpoint, but they can maintain E2F1 transcription, they can maintain the fibre length following replication stress
  this shows that E2F transcription is not just required but is also sufficient.

Question 60

what are the two mains roles of E2F upreg in cancer now thought to be?

Answer 60

normally E2F6 inhibits E2F, when there is replication stress, you upreg ATR, which upreg Chk1 which inhibits E2F6 and allows E2F to be transcribed. This allows E2F to trascribe the check point proteins and replication proteins to be maintained: arrest replication fork, stabilise stalled forks and resume replication after RS.
another is that oncogene induced replication stress is a result of E2F activity that stimulates proliferation.
- would make sense to do both to accommodate the other

Question 61

how could they test the role of E2F upreg in cancer?

Answer 61

they already had a cell line in which they could induce E2F dependent transcription to turn E2f-dependent transcription on in response to replication stress. They can combine this with the ability to induce c-myc (a potent oncogene) in the same cel line.
they found that if they induce E2F6, you see that this decreases the expression level of E2F-target CtIP.
if you activate the oncogene c-myc, you see a significant increase in E2F-dependent transcription, using CtIP as the marker. this is because increased rep stress and increased chekcpoint increase in E2F. If at the same time as c-myc you add E2F6, you see a decrease in this target.
so they validated this as a system of activating oncogenic activity with and without E2F dependent transcription.
then you can ask what is the role of E2F dependent transcription in ongene induced replication stress?

Question 62

how did they show that E2F prevent cancer DNA damage in the presence of replication stress? what about their tolerance to replication stress?

Answer 62

The first thing they looked at was DNA damage. in the presence of replication stress They found that if you apply E2F6 yo udont get an increase in DNA damage, then with c-myc yo get an increase, then with E2F6, you see that you get even more DNA damage. So E2f in the presence of c-myc can prevent DNA damage. you find that E2F also acts to promote cell viability in the presence of c-myc comapred to c-myc only without E2F6.
- they then did this both with DNA replication stress, and found that if you activate the oncogene in the presence of E2F (with E2F6 inhibition, you see that few cells experience replication induce DNA damage compared to in the presence of E2F6. The importnat point here is that these cells are still experiencing replication stress, but this does not results in DNA daamge in the resence of E2F. so this shows that E2F is required for the tolerance- not from the lowering ot the replicatin stress, just in the tolerance.

Question 63

how can the role of E2F in cancer be exploited?

Answer 63

• looking for cancer drugs that can impair the DNA damage repair pathway but this also harm healhty cells, but it has been shown that in cancer cells there is a high level of pertubation to DNA repair pathways anyway.
• there are cancer associated defects in DNA repair pathways: so if you use an anti cancer drug to target protein B, and protein A already has a cancer associated defect, then the cell will die. but this wont happen in the healthy cell because it has an alternative pathway to use.
  So they want to establish how is dna replication stress induced and they want to establish which proteins are most involved in DNA replication stress buffering, and they hope to target these the mechanisms so that more DNA replication stress occurs, or target the buffers which mean that DNA replication stress cannot be dealth with, by targetting these pathways, they can induce catastrophic genomic instability in cancer cells. EVen if it doesnt, it will increase genomic instabilty but will not affect genome stability in healthy cells. SO could be used as an addtional therpay in cancer therapy. -