n. Lecture (Feb. 13th Slide Deck)

Question 1

Describe fxns of the following ubiquitin-protein ligases
a) SCF - 1
b) APC/C - 2

Answer 1

a) degradation of phosphorylated Sic1 to activate S phase CDKs
b)
- degradation of securin to initiate anaphase
- degradation of B cyclin in G1 to allow loading of helicases on ORC

Question 2

Match the following
a) CDK1
b) CDK2
c) CDK4
1. Cyclin A
2. Cyclin B
3. Cyclin D
4. Cyclin E
i. G1/S
ii. S
iii. M

Answer 2

a) 2 + iii
b) 1 + 4 + ii
c) 3 + i

Question 3

Put the following cyclins in order according to the cell cycle
a) cyclin A
b) cyclin B
c) cyclin D
d) cyclin E

Answer 3

D -> E -> A -> B

Question 4

a) What does CAK stand for?
b) What does it regulate?
c) What is the purpose of this regulation?

Answer 4

a) CDK-activating kinase
b) phosphorylates Thr residues near the active site of CDKs
c) This activates the CDKs that are required for upcoming part of the cell cycle

Question 5

T or F - CAK activity only occurs when the enzyme activity of a certain CDK is needed in the cell cycle

Answer 5

F - its activity is constant throughout the cell cycle as it needs to phosphorylate it once the cyclin-CDK complex is formed

Question 6

a) what is the role of Cdc25 wrt the regulation of the cell cycle?
b) What is the result of this? - 2
c) which cell cycle transitions does this influence? - 2

Answer 6

a) it’s a phosphatase that removes the inhibitory phosphorylation from Tyr and Thr residues w/in CDKs
b) this activates the G1/S and mitotic CDKs
c) G1 to S and G2 to M

Question 7

Describe the 3 regulation step for the G1/S transition using the following terms; G1/S phase CDKs, S phase cyclin CDKs, S phase CDK inhibitor, SCF-proteasome, degrade, phosphorylate, synthesis

Answer 7

1. The G1/S phase CDKs phosphorylates the S phase CDK inhibior that is inhibiting the S phase CDKs
2. the SCF-proteasome recognizes and degrades the phosphorylated S-phase CDK inhibitor
3. the S-phase CDKs are now active allowing for the synthesis phase to proceed

Question 8

Fill in the following

Answer 8

blue = G1/S phase CDKs
yellow = S phase CDK inhibitor
green = S phase CDKs
pink = SCF-proteasome
orange = S phase CDKs
purple = DNA replication

Question 9

Describe what occurs in the following transition wrt regulation
a) metaphase to anaphase
b) exiting mitotic phase

Answer 9

a) APC/C proteasome degrades securin allowing the cleavage of cohesions
b) activation of Cdh1 and APC/C proteasome which degrades mitotic cyclins

Question 10

What is the restriction pt?

Answer 10

the start pt of the cell cycle (start of G1 phase)

Question 11

What is the significance of G0 (Gnot)?

Answer 11

this the were cell that never divide arrest

Question 12

Describe the 4 steps for control of the G1/S transition in S. cerevisiae using the following terms; Whi5, SBF, CLN1 gene, CLN2 gene, Cln3-CDKs, nutrients, Cln1-CDK, Cln2-CDK, budding, S phase, Spindle pole body duplication, Target of Rapamycin (TOR), rRNA, synthesis, transcription factor, inhibitor

Answer 12

1. TOR senses the presence of nutrients causing the stimulation of rRNA which results in the synthesis of Cln3-CDKs
2. Cln3-CDKs phosphorylate the inhibitor Whi5 causing it to disassociate from the transcription factor SBF activating it
3. SBF promotes the synthesis of Cln1-CDK or Cln2-CDKs from the CLN1 or CLN2 gene
4. Cln1-CDK/Cln2-CDK causes budding, the transition to the S phase, and spindle pole body duplication

Question 13

Fill in the following for S. cerevisiae

Answer 13

yellow = Whi5
green = SBF
blue = CLN1 or CLN2 gene
pink = Cln3-CDKs
orange = nutrients
purple = Cln1-CDKs + Cln2-CDKs
black = budding, S phase, spindle pole body duplication

Question 14

Describe the 4 steps for control of the G1/S transition in mammals using the following terms; Rb, E2F, Cyclin E gene, cyclin A gene, cyclin D, CDK4, CDK6, growth factors (GF), Cyclin E, Cyclin A, CDK2, S phase, centrosome duplication, synthesis, transcription factor, inhibitor

Answer 14

1. the presence of GFs stimulates the synthesis of cyclin D which stimulates the expression of CDK4 and CDK6
2. CDK4 and 6 phosphorylate the inhibitor Rb causing it to disassociate from the transcription factor E2F activating it
3. E2F stimulates the synthesis of cyclin E and cyclin A which stimulates the expression of CDK2
4. CDK2 causes transition to S phase and centrosome duplication

Question 15

Fill in the following for mammals

Answer 15

yellow = Rb
green = E2F
blue = Cyclin E or cyclin A gene
pink = cyclin D + CDK4/6
orange = growth factors
purple = Cyclin E/A + CDK2
black = S phase + Centrosome duplication

Question 16

Describe the 4 steps for the onset of S phase in S. cerevisiae using the following terms; G1/S cyclin, CDK, G1/S cyclin-CDK complex, S phase cyclin-CDK complex, Sic1, phosphorylate, degrade

Answer 16

1. G1/S cyclin binds to CDK activating it
2. the G1/S cyclin-CDK complex phosphorylates the inhibitor Sic1
3. Sic1 falls off and gets degraded activating the S phase cycline-CDK complex
4. DNA replication takes place

Question 17

As see know Sic1 is an inhibitory protein that prevents the transition from G1 to S phase. In order to get rid of this inhibitor it is required to phosphorylate it. But why are there so many phosphorylation sites?

Answer 17

This speeds up the transition as it is easier to recognize and degrade Sic1 when it has been phosphorylated 6 times as opposed to less times

Question 18

What does this graph show wrt the Sic1 protein

Answer 18

Sic1 protein being phosphorylated at all its different sites at once

Question 19

What does this graph show wrt the Sic1 protein

Answer 19

Sic1 protein being phosphorylated at each of its phosphorylated sites one at a time

Question 20

Describe the following proteins used in the initaiton of DNA replicatoin
a) ORC
b) Cdc6
c) Cdt1
d) MCM-helicase
e) pre-replication complex

Answer 20

a) origin recognition complex = the complex that docks on the DNA in order to indicate where replication will start
b) a loader that associates w/ the ORC at low [CDK]
c) a loader that associates w/ the ORC at low [CDK]
d) minichromosomal maintenance helicase = a helicase that associates on either side of the ORC ready to unwind the DNA
e) formation of all the proteins (a to d)

Question 21

When is pre-RC (pre-replication complex) formed?

Answer 21

early G1 phase when [CDK] is low

Question 22

Describe the steps for the initiation of DNA replication using the following terms; S-phase cyclin, inhibitor, degrade, kinase, DDK, S phase CDK, phosphorylate, Cdc6, Cdt1, MCM helicase, replication fork, CDC45, Sld2, GINS, protein, elongation

Answer 22

1. S-phase cyclins are synthesized while the inhibitors of those S-phase cyclins are degraded
2. the kinases DDK and S phase CDK phosphorylate Cdc6 + Cdt1 causing them to degrade
3. DDK and S phase CDK phosphorylates the MCM helicase causing the production of the replication fork (ORC falls off)
4. CDC45 and Sld2 recruits GINS resulting in all of these proteins associating w/ the MCM helicases
5. elongation takes place

Question 23

a) what are cohesion rings?
b) where are they located?
c) What are the 2 main regions of a cohesion ring?

Answer 23

a) a ring protein that keeps sister chromatids together
b) at the centromere
c) Smc1 and Smc3

Question 24

T or F - during DNA synthesis Smc1 is methylated, leading to encirclement of the replicated sister chromatids by a cohesion ring

Answer 24

F - Smc3, acetylated

Question 25

Describe the following proteins wrt the cohesion ring a) Smc1 and Smc3 b) Polo an Aurora c) APC

Answer 25

a) the two protein monomers that dimerize and make up the cohesion ring b) kinases that phosphorylate cohesion ring c) a ubiquitin-protein ligase that recognizes the phosphorylated cohesion ring and degrades it

Question 26

At which phase do cohesion rings degrade? a) G1 b) S c) G2 d) M e) anaphase

Answer 26

e

Question 27

At which phase are cohesion rings synthesized? a) G1 b) S c) G2 d) M e) anaphase

Answer 27

a

Question 28

What acetylates the cohesion ring?

Answer 28

CoA = cohesion acylation transferase

Question 29

a) what is the purpose of checkpoints? b) What are the 3 components that make up a checkpoint pathway? c) What are the 2 things that can cause the checkpoint pathway to arrest the cell cycle progression?

Answer 29

a) to ensure that the next stage of the cell cycle doesn't start until the prev one is completed b) 1. event sensors 2. a signaling pathway - conveys that event sensor stim 3. effectors that cause the pause c) DNA damage or improper spindle assembly

Question 30

What is the purpose of the presence of nutrients/growth factor for G1/S transtion?

Answer 30

to ensure that the cell has enough proteins and NRG to go through the cell cycle

Question 31

How will the G1/S checkpoint react to the following sit a) lack of nutrient/growth factors b) abundance of nutrients/growth factors c) damaged DNA

Answer 31

a) remain halted until its sufficient b) G1/S transition will take place c) G1/S point will be inactivated preventing further progress until repairs take place or apoptosis of cell

Question 32

Describe the 2 things that are important for the following models wrt transitioning from G1 to S phase a) budding yeast b) mammalian cells

Answer 32

a) - nutrients that stimulates the synthesis of Cln3 - TOR (Target of Rapamycin) that regulates the activity of ribosomes and rRNA synthesis - ensuring that the cell can synthesize all the proteins needed for the cell cycle to progress b) - growth factors stimulate the synthesis of Cyclin D which phosphorylates Rb - TOR regulates the activity of CDKs and the synthesis of various genes needed to ensure cell cycle can progress

Question 33

T or F - TOR is only present in yeast cells

Answer 33

F - they are present in mammalian cells as well they just do slightly different things

Question 34

Indicate which kinase (ATM or ATR) is activated by the following types of DNA damage a) Stalled replication fork b) DNA mismatches c) double-strand breaks d) Nucleotide damage

Answer 34

a) ATR b) ATR c) ATM d) ATR

Question 35

Which of the following kinases are activated by ATM and/or ATR a) Chk2 b) Chk1

Answer 35

a) ATM and ATR b) ATM

Question 36

What are the 3 things that activated Chk1 and Chk2 do as a result of DNA damage

Answer 36

1. initiate the repair of DNA 2. phosphorylate cdc25 which inhibits CDKs in order to cause in pause in the cell cycle progression 3. activates p53 which either stimulates p21 which arrests the cell cycle (when at low levels) or triggers apoptosis (when at high levels)

Question 37

Draw the flow chart that involved the response to DNA damage using the following terms; Cdc25, Repair, ATM, p21, DNA damage, Chk1, ATR, p53, Chk2, apoptosis, CDK

Answer 37

Question 38

Draw the response to DNA damage during the G1 phase

Answer 38

Question 39

Draw the response to DNA damage during the G1/S phase checkpt

Answer 39

Question 40

Draw the response to DNA damage during S phase checkpt

Answer 40

Question 41

Draw the response to DNA damage during the M phase checkpt

Answer 41