Cell Cycle Flashcards
Explain the enter S checkpoint. (3 mechanisms)
Hint: talk about Rb’s effect on E2F, ATM/ATR’s effect on Mdm2, and Myc + E2F’s effect on Mdm2)
In G1 Rb normally binds to E2F, inhibiting it.
When active cdk’s phosphorylate Rb, E2F is free to promote the txn of S-phase genes as a txn factor.
Double strand breaks during G1 prompt the activation of ATM and ATR kinases. The ATM/ATR kinases then phosphorylate Mdm2 and p53, prompting them to dissociate. p53 is now active and serves as a txn factor for p21, which inhibits G1/S- and S-cdk. The G1/S- and S-cdk’s are now unable to phosphorylate Rb, and cell remains in G1/G0.
Excess Myc and E2F signaling can upregulate Arf1. Arf1 inhibits Mdm2’s binding p53. More p53 –> more txn of waf1 gene –> more p21
Explain the enter M checkpoint.
CAK phosphorylates M-cdk at the activation site.
Wee-1 kinases phosphorylate M-cdk at the inhibitory site.
Cdk25 dephosphorylate’s the inhibitory site.
Activated cyclin A’s then offer positive feedback by driving the phosphorylation of Cdc25 and inhibiting Wee1.
DNA damage results in the increased phosphorylation of the inhibitory site.
Explain the exit M checkpoint.
Ensures all chromatids are attached to microtubules (spindle fibers) before progressing to anaphase.
Normally, following metaphase, APC is activated. It then inhibits securin. This allows separase to drive the cell into anaphase.
When kinetochores are unjoined (eg. disconnected from EITHER chromosomes or microtubules), they release Cdc20. This inhibits Apc/c and prevents anaphase from happening., Apc/c is an E3 ligase that ubiquitinates cyclin B’s.
The cell is in G1. The extracellular and intracellular environments are favorable. Describe the changes that activate G1-cdk and allow the transition to S-phase.
The inactivation G1-cdk is dephosporylated.
G1/S cyclin and S cyclin are sythesized.
The cdk can now bind G1/S-cyclin and does so in equilibrium.
Some of the cdk also binds S-cyclin.
When enough of it binds S-cyclin, S-cdk predominates, and cell enters S-phase.
Define and explain the methods that can be used to analyze cell cycle
The cell cycle can be observed via microscopy.
3H-thymidine (less harsh) or BrdU can be incorporated into the strand during S phase. Can then assay to find whether cells were actively replicating.
Fluorochrome PI intercalates in nucleic acids. Fluorescence increases 50 fold.
Flow cytometry - DNA-binds fluorescent dyes, which quantitatively double during S phase. This can allow you to determine the lengths of the four phases.
Hydroxyurea inhibits DNA replication
Colcichine (anti-microtubule) stops mitosis
Define the features of the cell cycle control system (6)
1 Fixed amount of time for each phase
2 Initiates events in the correct order
3 Event must occur only once per cycle
4 Switches trigger events in an irreversible fashion
5 Backup mechanisms, in case parts of system malfunction
6 Adaptability so that the systems behavior can be modified to suit certain cell types or environmental conditions
Define RhoA and clarify its role in cytokinesis
RhoA is a small GTPase. Activated RhoA (with GTP attached) promotes the assembly of actin filaments in the contractile ring.
What is the function of the cell cycle?
To allow an orderly pattern of DNA replication and DNA separation that minimizes errors and allows the cell time to grow and synthesize a sufficient amount of relevant proteins.
How does the cell stop continuation of the cell cylce when the extracellular environment is unfavorable?
Growth factors not present at high concentrations, so no growth factor signal is generated through MAPK pathway. CKI levels high inhibiting G1-Cdk activity. Cyclins not synthesized. Wee1-like kinases active.
What are two mechanisms of positive feedback at the mitosis checkpoint?
Active M-cdk’s inhibit Wee-1 and activate (via phosphorylation) Cdc25.
Which cyclin peaks at the start of mitosis?
cyclin B
Which cyclin peaks at the onset of S phase
cyclin E
Which cyclin peaks at the start of G2?
cyclin A
When do cyclins B, A and E appear?
Cyclin E- peaks at S phase
Cyclin A- peaks at G2 phase
Cyclin B- peaks at M phase
(like BAE but backwards)
