Cell Cycle and Control

Question 1

DNA replication occurs in ___ phase

2 daughter cells are generated in ___ phase

Answer 1

S phase = replication

M phase = 2 daughter cells generated (mitosis + cytokinesis)

Question 2

interphase = __ + ___ + ___

what is purpose of interphase

Answer 2

interphase = G1 + S + G2

time of delay to accumulate mass, monitor intra/extra-conditions, point of regulation

Question 3

explain goals of restriction point of G1

Answer 3

“should I invest all this energy in replication?”

measures favorably of environment
if passes, cell is COMMITTED

(if environmental signals are unfavorable AFTER restriction point, oh well)

Question 4

senescence

Answer 4

cells don’t pass the bar at G1 restriction point, hang out in G0 until conditions become more favorable

(after passing, cell is committed)

Question 5

4 goals of cell cycle control

Answer 5

1. turns on at specific times (clockwork)
2. events are in correct order
3. each event is only 1x time
4. on/off switches that trigger events in complete and irreversible fashion

Question 6

major enzymes of cell cycle control

Answer 6

cyclin-dependent kinases (Cdks) - activity of these increase or decrease during different phases

phosphorylation of target proteins initiatives or regulates cell cycle events

DEPENDENT ON CYCLINS (different cyclins for different phases)

Question 7

4 classes of Cdks?

Answer 7

1. G1 Cdk - promotes passage through restriction point (cyclin D)
2. G1/S Cdk - commits cell to replication (cyclin E)
3. S Cdk - initiates replication (cyclin A)
4. M Cdk - promotes mitosis (cyclin B)

Question 8

how does S-Cdk also prevent re-replication?

Answer 8

phosphorylation of Cdc6 by S-Cdk causes Cdc6 to dissociate from ORC (pre-RC disassembled)

dissociation/phosphorylation of Cdc6 causes its degradation

S-Cdk also phosphorylates Mcm (helicase) proteins to cause export from nucleus

Question 9

describe steps of entry into S phase

Answer 9

1. ORC binds origins and recruits regulatory proteins
2. Cdc6 increases transiently in early G1, binds ORCs —> Mcm (helicases) recruited (ORC + Cdc6 + Mcm = pre-replicative complex)
3. S-cyclin transcription activated in late G1, S cyclin-Cdk complex phosphorylates DNA pol (pre-RC activated)
4. S-Cdk assembles DNA pol/replication machinery at origins, activates Mcm (helicases) —> replication is triggered

Question 10

how does M-Cdk prevent replication occurring twice?

Answer 10

phosphorylates Cdc6 and Mcm (helicase) proteins

*keep in mind S-Cdk activity remains high during G2 and mitosis to keep Cdc6 phosphorylated/inactivated

[at end of mitosis, all Cdk activity is zeroed so Cdc6 and Mcm can be DEphosphorylated to allow for pre-Rc assembly again]

Question 11

regulation of M-Cdk is controlled by 3 proteins:

Answer 11

1. M cyclin transiently increases during G2 and M phases (transcribed)
2. CAK (Cdk activating kinase)
3. Wee1 (Cdk inhibitory kinase, inhibitory phosphorylation)

Question 12

M-Cdk is “poised” for activation - what needs to be done to activate it?

Answer 12

Cdc25 (phosphatase) removes inhibitory phosphate (Wee1, Cdk inhibitory kinase)

***not all Cdc’s are phosphatase - they each have unique mechanisms and roles (don’t group them)

Question 13

M-Cdk phosphorylates proteins responsible for: (3 things)

Answer 13

1. assembly of spindle for chromosome segregation
2. chromosome condensation
3. breakdown of nuclear envelope

Question 14

describe the feedback loops (2) to increase M-Cdk activity

Answer 14

1. M-Cdk inhibits Wee1 (inhibitory kinase of M-Cdk)
2. M-Cdk phosphorylates more Cdc25 (phosphatase which removes Wee1)

Question 15

exit from mitosis requires inactivation of M-Cdk which occurs primarily through _____

Answer 15

ubiquination (degradation)

M-Cdk regulates its own degradation

Question 16

during what phase of the cell cycle is Cdk activity ABSENT, and what mechanisms ensure this?

Answer 16

G1 - NO Cdk activity, via:

1. ubiquination of Cdks
2. Cyclin Kinase Inhibitor (CKI) accumulates
3. decreased cyclin transcription via Rb (retinoblastoma) protein and E2F transcription factor (Rb INHIBITS E2F)

Question 17

what protein and transcription factor are needed to decrease cyclin transcription during G1 (to ensure absence of Cdk activity)?

Answer 17

Rb (retinoblastoma) protein and E2F transcription factor

E2F function is controlled by Rb protein —> during G1, Rb binds/blocks E2F (G1/S and S cyclins NOT transcribed)

extracellular signal to divide —> G1-Cdk accumulates and phosphorylates/inactivates Rb (loses affinity for E2F) —> G1/S and S cyclins transcribed

Question 18

how do CDK inhibitor proteins (CKIs) such as p27 work?

Answer 18

binds active cyclin-Cdk complex —> inactivated

Question 19

why does loss of both copies of Rb gene cause a cancer, retinoblastoma?

Answer 19

Rb’s job is to bind/block E2F transcription factor for cyclin transcription

without it, cyclins are transcribed, which continue to activate Cdks, and cell cycle continues unregulated

Question 20

describe the feedback loop for Rb protein (3 ways)

Answer 20

when E2F is released from Rb, it increases its own expression

E2F expression leads to production of G1/S-Cdk and S-Cdk —> G1-Cdk phosphorylates Rb (inactivates, MORE E2F released)

increased G1/S-Cdk and S-Cdk enhance phosphorylation of ubiquitin ligases and CKIs, leading to their destruction in proteosome — G1/S-Cdk and S-Cdk activated

Question 21

what is the purpose of G2 checkpoint

Answer 21

prevents mitosis when there is DNA damage so it’s not passed on

damaged DNA sends signal that blocks Cdc25 activity (phosphates that removes Wee1 inhibitory kinase) —> M-Cdk remains phosphorylated and inactive

Question 22

what is the purpose of the G1 checkpoint and how does it work?

Answer 22

G1 checkpoint prevents progression into S (replication) phase

inhibits activation of G1/S-Cdk and S-Cdk complexes - controlled by p53

p53: stimulates expression of several genes including CKI and p21

CKI then binds to G1/S-Cdk and S-Cdk to inactive them

Question 23

how does p53 work to check cell cycle in G1 checkpoint?

Answer 23

during G1 DNA damage checkpoint, stimulates expression of several genes including CKI and p21

CKI binds to G1/S-Cdk and S-Cdk to inactive them

entry into S phase prevented

Question 24

Fill in:

At the beginning of __ phase, ____ activates a ubiquitin-ligase that then results in the degradation of _____

Answer 24

At the beginning of M phase, M-Cdk activates a ubiquitin-ligase that then results in the degradation of M-Cdk

regulates itself

Question 25

Which of these feedback mechanisms is NOT regulated by M-Cdk?
a. phosphorylation and further activation of Cdc25
b. activation of APC ubiquitin-ligase
c. Inhibition of Wee1
d. Activation of Wee1

Answer 25

M-Cdk DOES regulate these:

a. phosphorylation and further activation of Cdc25
b. activation of APC ubiquitin-ligase
c. Inhibition of Wee1

does NOT activate Wee1

Question 26

Which of these is not an immediate effect of Rb deletion?
a. increased levels of E2F
b. increased levels of S-Cdk
c. re-entry into G1
d. decreased expression of Cdc25

Answer 26

these are immediate effects of Rb deletion:
a. increased E2F
b. increased S-Cdk
c. re-entry into G1

Rb deletion would not inherently cause decreased expression of Cdc25