The cell cycle Flashcards
Define the cell cycle
The process by which a cell duplicates its contents in order to produce two daughter cells
What 2 major processes does the cell cycle involve?
- DNA replication
- Accurate chromosome segragation
What occurs during the S phase
Replication of chromosomes
What occurs during the M phase
Segregation of chromosomes (mitosis) before the cell divides (cytokinesis)
Name the 5 stages of mitosis in the order that they occur
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
What key change occurs at the metaphase to anaphase transition?
Sister chromatid cohesion (formed during S phase) is lost and the sister chromatids are pulled appart
What are the 4 main stages of the cell cycle in order?
G1 phase
S phase
G2 phase
M phase
What is the function of the gap (G) stages?
- allow cell to grow
- prepare for S and M phases
- monitoring of internal and external cell environment
What occurs when conditions are not favourable at the G1-S transition?
Cells enter specialised resting state referred to as Gzero
How is cell cycle progression controlled?
- 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
Why is budding yeast a useful model for the study of the cell cycle?
- Generation time of under 2 hours
- Simple to grow in the lab
- Very amenable to genetic manipulation
- Can be maintained in haploid/diploid form
What is the advantage of using haploid organisms for studies?
- 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
Why is xenopus laevis a good model for cell cycle investigations?
- Egg extracts and sperm can be mixed to recapitulate early cell cycle events
- Egg extracts can be analysed biochemically by depletion of individual factors
What are the 3 main regulatory points in the cell cycle?
G1-S
G2-M
Metaphase-anaphase
How is the progression through the cell cycle regulated?
CDK-cyclin complexes
What are CDK-cyclin complexes composed of?
- catalytic subunit containing protein kinases (CDKs) which are present at a constant level
- regulatory subunits - cyclins which undergo synthesis and destruction
What are the 3 essential classes of cyclins and their roles?
- 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
Appart from activating CDK enzymes, what is the other role of cyclins?
Confer substrate specificity
Describe an additional method of cell cycle regulation
Some substrates are only available at some points during the cell cycle
How are cyclin dependant kinases activated?
- 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
How is M-CDK activity regulated?
- 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
How is M-CDK activity amplified during the G2-M transition?
Through positive feedback loops
How do CDK inhibitor proteins work?
e.g p27
distorts active site and partially blocks the ATP-binding site
How is the metaphase-anaphase transition triggered?
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
How does APC/C carry out regulated proteolysis?
- Becomes covalently attached to a lysine side chain of the target protein
- Polyubiquitin chains are then formed by linking more ubiquitin chains via Lys48
How does APC/C interact with the proteasome?
- 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
How is APC/C activated?
mid-mitosis - Cdc20
late mitosis/ early G1 - Cdh1
What needs to happen after S/M-CDK activity has been abolished in order to complete mitosis & cytokinesis?
Proteins previously phosphorylated during the S phase to early mitosis need to be dephosphorylated before mitosis/cytokinesis can be completed
Why is the protein securin targetted by APC/C action?
- 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
What is SCF?
A ubiquitin ligase that functions during S phase
What is the function of SCF?
targets various CKIs for destruction in late G1 and is responsible for G1/S cyclin destruction in early S-phase
How is SCF regulated differently to APC/C?
SCF is always presents but requires substrates in order to be phosphorylated
What is required for the initiation of chromosome replication?
- triggered by an increase in S-Cdk activity
- Assembly of pre-RC complexes occurs from late mitosis-early G1 when CDK activity is low
How is DNA replication regulated so that it only occurs once per cell cycle?
- 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
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?
MCDK - prophase, prometaphase, metaphase
APC/C - anaphase, telophase, cytokinesis
Which events are triggered by M-CDK activity?
- chromosome condensation (phosphorylates condensin subunits)
- spindle assembly
- nuclear envelope breakdown
- rearrangement of the actin cytoskeleton
- rearrangement of the Golgi apparatus
How can APC/C activation be inhibited?
- 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
