PAUSE - CELL CYCLE 2

Question 1

when do the different checkpoints of the cell cycle happen?

Answer 1

G1 checkpoint: is the environment favorable
G2 checkpoint: is all DNA replicated, is the environment favorable?
Metaphase checkpoint: are all chromosomes attached to the spindles?

Question 2

what do checkpoints do?

Answer 2

negative signals that can stop the cell cycle
not required
no checkpoints = no control = cancer

Question 3

what is the key complex of the cell cycle?

Answer 3

cyclin + cyclin dependent kinase complex
without cyclin cdk is inactive

Question 4

how does the amount of cyclins change through the cell cycle?

Answer 4

goes up before S phase, triggers DNA replication by phosphorylation

Question 5

how does cdk work?

Answer 5

associate successively with different cyclins to trigger the different events of the cycle

Question 6

how is cdk activity terminated?

Answer 6

by degradation

Question 7

what does the S-cdk complex do?

Answer 7

trigger DNA replication machinery

Question 8

what does the M-cdk complex do?

Answer 8

trigger mitosis machinery

Question 9

what is specific and what is not?

Answer 9

cyclin is specific
cdk is not

Question 10

how is cdk activated?

Answer 10

1. no cyclin bound
   active site is blocked by T-loop
2. cyclin binds and T loop moves out of active site, resulting in partial activation of the cdk
3. phosphorylation of cdk by cdk-activating kinase (CAK) at a threonine residue in the T loop further activates the enzyme by changing shape of T loop
   improves ability of enzyme to bind to protein substrates

Question 11

how does the cyclin-cdk complex switch from active to inactive?

Answer 11

turned off when kinase Wee1 phosphorylated two closely spaced sites above the active site
removal of those P by phosphatase Cdc25 activates the complex chain

Question 12

how is a cyclin-Cdk complex inhibited by a CKI?

Answer 12

CKI p27 binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk
inserts into the ATP binding site, further inhibiting the enzyme activity

Question 13

what does the phosphorylation of a CKI lead to?

Answer 13

allows the protein to be recognised by SCF which is always active
with the help of E1 and E2, SCF is a ubiquitin ligase onto the CKI protein
recognised and degraded by proteasome

Question 14

how is proteolysis controlled by APC?

Answer 14

M cyclin ubiquitylation is performed by APC
activated in late mitosis by the addition of an activating subunit to the complex
SCF and APC contain binding sites that recognise specific amino acid sequences to the target protein

Question 15

how did the fusion of cells in different phases provide evidence for a rereplication block?

Answer 15

fuse a cell in S phase with a cell in G1 phase and the cell entered S phase
S phase was fused with G2 phase cell,
nothing happens, G2 remains in G2
G1 cell fused with G2, nothing happens
the factors that drive DNA replication are present in S phase but no longer in G2

Question 16

how is DNA replication initiated once per cycle?

Answer 16

1. the ORC (origin recognition complex) is associated with the replication origin throughout the cell cycle
2. in early G1, Cdc6 associated with ORC
3. aided by Cdc6, Mcm ring complexes assemble on adjacent DNA, resulting in formation of the pre-replicative complex
4. the S-Cdk (with assistance from another protein kinase) triggers origin firing
5. assembly of DNA polymerase and other replication proteins and activating the Mcm proteins rings to migrate along DNA strands as DNA helicases
6. S-Cdk also blocks re replication by causing dissociation of Cdc6 from origin, its degradation, and the export of all excess Mcm out of nucleus
7. Cdc6 and Mcm cannot return to reset an ORC containing origin for another round of DNA replication until M-Cdk has been inactivated at the end of mitosis

Question 17

how does the activation of M-Cdk work?

Answer 17

1. Cdk1 associated with M-cyclin as the levels as the levels of M-cyclin rise
2. resulting M-Cdk complex is phosphorylated on an activating site by the CAK and on a pair of inhibitory sites by Wee1 kinase
3. resulting inactive M-Cdk complex is activated at the end of G2 by phosphatase Cdc25
4. Cdc25 is stimulated in part by polo kinase
5. Cdc25 is further stimulated by active M-Cdk resulting in positive feedback
6. this is enhanced by the ability of M-Cdk to inhibit Wee1

Question 18

how do concentrations of Cdk change during the cell cycle, and how do they compare cyclin concentrations?

Answer 18

cdk concentrations do not change and exceed cyclin amounts

Question 19

how do the different concentrations of cyclins change throughout the cell cycle?

Answer 19

1. In late G1, rising G1/s-cyclin levels lead to the formatoon of G1/S-Cdk complexes
2. trigger progression through the start transition.
3. S-Cdk complexes form at the start of S phase and trigger DNA replication, as well as some early mitotic events.
4. M-Cdk complexes form during G2 but are held in an inactive state;
5. they are activated at the end of G2 and trigger entry into mitosis at the G2/M transition.
6. A separate regulatory protein complex, the ApC/C, initiates the metaphase-to-anaphase transition, as we discuss later.