The cell cycle

Question 1

Define the cell cycle

Answer 1

The process by which a cell duplicates its contents in order to produce two daughter cells

Question 2

What 2 major processes does the cell cycle involve?

Answer 2

• DNA replication

- Accurate chromosome segragation

Question 3

What occurs during the S phase

Answer 3

Replication of chromosomes

Question 4

What occurs during the M phase

Answer 4

Segregation of chromosomes (mitosis) before the cell divides (cytokinesis)

Question 5

Name the 5 stages of mitosis in the order that they occur

Answer 5

1. Prophase
2. Prometaphase
3. Metaphase
4. Anaphase
5. Telophase

Question 6

What key change occurs at the metaphase to anaphase transition?

Answer 6

Sister chromatid cohesion (formed during S phase) is lost and the sister chromatids are pulled appart

Question 7

What are the 4 main stages of the cell cycle in order?

Answer 7

G1 phase
S phase
G2 phase
M phase

Question 8

What is the function of the gap (G) stages?

Answer 8

• allow cell to grow
• prepare for S and M phases
• monitoring of internal and external cell environment

Question 9

What occurs when conditions are not favourable at the G1-S transition?

Answer 9

Cells enter specialised resting state referred to as Gzero

Question 10

How is cell cycle progression controlled?

Answer 10

• complex network of regulatory interactions
• include ordered biochemical switches and checkpoints that ensure the next stage has not started before all earlier things are completed
• monitoring of outside signals in response to developmental/environmental cues

Question 11

Why is budding yeast a useful model for the study of the cell cycle?

Answer 11

• Generation time of under 2 hours
• Simple to grow in the lab
• Very amenable to genetic manipulation
• Can be maintained in haploid/diploid form

Question 12

What is the advantage of using haploid organisms for studies?

Answer 12

• Genes can be mutated to investigate function without interference from a second copy
• However can only use conditional lethal mutants, as inactivation of essential genes would cause organism death

Question 13

Why is xenopus laevis a good model for cell cycle investigations?

Answer 13

• Egg extracts and sperm can be mixed to recapitulate early cell cycle events
• Egg extracts can be analysed biochemically by depletion of individual factors

Question 14

What are the 3 main regulatory points in the cell cycle?

Answer 14

G1-S
G2-M
Metaphase-anaphase

Question 15

How is the progression through the cell cycle regulated?

Answer 15

CDK-cyclin complexes

Question 16

What are CDK-cyclin complexes composed of?

Answer 16

• catalytic subunit containing protein kinases (CDKs) which are present at a constant level
• regulatory subunits - cyclins which undergo synthesis and destruction

Question 17

What are the 3 essential classes of cyclins and their roles?

Answer 17

• G1/S cyclins which activate in late G1, committing to S-phase entry (start)
• S phase cyclins interact with CDKs after the ‘start’ to initiate DNA replication
• M phase cyclins activate CDKs to drive entry into mitosis

Question 18

Appart from activating CDK enzymes, what is the other role of cyclins?

Answer 18

Confer substrate specificity

Question 19

Describe an additional method of cell cycle regulation

Answer 19

Some substrates are only available at some points during the cell cycle

Question 20

How are cyclin dependant kinases activated?

Answer 20

• Cyclin binds to T-loop on ATP molecules in kinase, causing partial activation
• kinase CAK then phosphorylates serine/threonine in the T-loop in order to further enhance activity

Question 21

How is M-CDK activity regulated?

Answer 21

• Wee1 protein kinase phosphorylates Cdk1 on two neighbouring amino acids to the active site causing inhibition
• Cdc25 phosphoprotein phosphatase removes phosphates in order to relieve inhibition

Question 22

How is M-CDK activity amplified during the G2-M transition?

Answer 22

Through positive feedback loops

Question 23

How do CDK inhibitor proteins work?

Answer 23

e.g p27

distorts active site and partially blocks the ATP-binding site

Question 24

How is the metaphase-anaphase transition triggered?

Answer 24

Via regulated proteolysis

• Protein destruction triggered by catalysed by a ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C)
• S- and M-cyclins are a major target of APC/C

Question 25

How does APC/C carry out regulated proteolysis?

Answer 25

- Becomes covalently attached to a lysine side chain of the target protein - Polyubiquitin chains are then formed by linking more ubiquitin chains via Lys48

Question 26

How does APC/C interact with the proteasome?

Answer 26

- Has 3 domains: ring, central cylinder and cap - Unfoldase at the ring unfolds the target protein, ubiquitins are then cleaved off and then cyclins are broken down at the central cylinder by protease

Question 27

How is APC/C activated?

Answer 27

mid-mitosis - Cdc20 | late mitosis/ early G1 - Cdh1

Question 28

What needs to happen after S/M-CDK activity has been abolished in order to complete mitosis & cytokinesis?

Answer 28

Proteins previously phosphorylated during the S phase to early mitosis need to be dephosphorylated before mitosis/cytokinesis can be completed

Question 29

Why is the protein securin targetted by APC/C action?

Answer 29

- Securin inhibits the activity of the protease separase - When degraded at the end of metaphase, separase can then cleave off subunits of protein cohesin resulting in sister chromatid separation at anaphase

Question 30

What is SCF?

Answer 30

A ubiquitin ligase that functions during S phase

Question 31

What is the function of SCF?

Answer 31

targets various CKIs for destruction in late G1 and is responsible for G1/S cyclin destruction in early S-phase

Question 32

How is SCF regulated differently to APC/C?

Answer 32

SCF is always presents but requires substrates in order to be phosphorylated

Question 33

What is required for the initiation of chromosome replication?

Answer 33

- triggered by an increase in S-Cdk activity | - Assembly of pre-RC complexes occurs from late mitosis-early G1 when CDK activity is low

Question 34

How is DNA replication regulated so that it only occurs once per cell cycle?

Answer 34

- S-CDK phosphorylates 2 proteins (Sld2 and Sld3) in order to facilitate the assembly of the CMG complex (replicative helicase in eukaryotes) - CMG is then fully activated using protein kinase DDK - Mechanisms such as protein phosphorylation, degredation, nuclear exclusion and inhibitor binding then prevent pre-RC complexes from reassembling until the next cell cycle

Question 35

Overall, which stages in the cell cycle are caused by M-CDK activity and which stages are caused by APC/C destruction of securin and cyclins?

Answer 35

MCDK - prophase, prometaphase, metaphase | APC/C - anaphase, telophase, cytokinesis

Question 36

Which events are triggered by M-CDK activity?

Answer 36

- chromosome condensation (phosphorylates condensin subunits) - spindle assembly - nuclear envelope breakdown - rearrangement of the actin cytoskeleton - rearrangement of the Golgi apparatus

Question 37

How can APC/C activation be inhibited?

Answer 37

- If chromosome fails to attach to spindle it will bind to the Mad2 protein which then inhibits Cdc20 which goes on to inhibit APC/C