Chapter 13: THE EUKARYOTIC CELL CYCLE

Dividing the cell cycle:

∑ Phases - Order and events (13-1).

∑ Regulation - progression through cell cycle phases is controlled by cyclin dependent kinases (Cdks) (13-2); Cdks are regulated by cyclin; cyclins determine which proteins Cdks will phosphorylate; there are several Cdks and cyclins that function during different stages of the cell cycle; G1 Cdk act after the cell is stimulated to divide; S Cdk triggers replication of the chromosomes after an S Cdk inhibitor is degraded by G1 Cdk; mitotic Cdk promotes chromosome condensation, spindle formation, metaphase alignment and activate the anaphase-promoting complex (APC); APC allows chromosomes to segregate and decondense and the nuclear envelope to reform; G1 Cdk inactivates APC; the cell cycle is irreversable; the cell cycle is stimulated by mitogens before surpassing the restriction point.

Regulating entry and exit from mitosis:

∑ Xenopus oocyte maturation - Arrested in G2 (13-5); progesterone induces entry into meiosis I up to metaphase of meiosis II; MPF can mediate maturation without progesterone (13-5); MPF activity is high during mitosis and low during interphase (13-6); MPF = kinase composed of a regulatory and catalytic subunit.

∑ Cyclins - Cyclin B is the regulatory subunit of MPF; cyclin B protein levels fluctuate throughout the cell cycle; cytoplasmic extracts from Xenopus eggs have all components necessary for the cell cycle; the cell cycle can occur in vitro when chromatin is added to egg extracts (13-7a); MPF activity is cyclin B mRNA and protein dependent (13-7b,c,d), and induces exit from mitosis; each cyclin contains a destruction box which leads to polyubiquitination and degradation in the proteosome (13-8); destruction is self promoted by phosphorylation of APC (13-9).

Cell cycle control in fission yeast:

∑ Fission yeast cell cycle (13-10); cdc and wee mutant phenotypes (13-11).

∑ Yeast cell cycle mutants - cdc2 is a protein kinase that corresponds to the catalytic subunit of MPF; cdc2 and the catalytic subunit of MPF are very similar in sequence; cdc13 is the fission yeast homologue of cyclin; cdc2 and cdc13 bind to form MPF.

∑ Regulating fission yeast MPF - The cdc25 and wee1 mutants have phenotypes that suggest defects in entry to mitosis (13-12); MPF is regulated by wee1, CAK and cdc25 (13-13); wee1 inactivates MPF by phosphorylating Y15, CAK phosphorylates inactive MPF at T161, and cdc25 activates MPF by removing phosphate on Y15 (25-22); substrate specificity and binding come from cdc13 + cdc2.

Regulation of mitotic events:

∑ Early events in mitosis include chromosome condensation, spindle formation, nuclear envelope breakdown; early events mediated by MPF dependent protein phosphorylation; lamin structure and nuclear envelope breakdown via phosphorylation of lamins (13-15); chromosome condensation controlled by condensin, which winds chromosomes into supercoils upon phophorylation; spindle formation and Golgi breakdown are also controlled by phosphorylation.

∑ Late events include chromosome segregation and movement to spindle poles, dismantling the spindle, chromosome decondensation, nuclear envelope and Golgi reformation; late events mediated by protein destruction or dephosphorylation; cohesins hold centromeres of sister chromosomes together, and are inactivated by the destruction of anaphase inhibitor by the APC (13-18); control of entry into anaphase and exit from mitosis (13-19); degradation of MPF enables dephosphorylation of lamins by phosphatases, which allows for the reformation of the nuclear envelope (13-20).

Cell cycle control in budding yeast:

∑ Budding yeast cell cycle (13-22); yeast cells must grow to reach a critical size to commit to entering S phase - a threshold called START.

∑ Budding yeast cdc28 is a Cdk similar to fission yeast cdc2, except fission yeast lacking cdc2 arrest at G₂ (entry to mitosis) and budding yeast lacking cdc28 arrest at G₁ (entry to S phase); interaction with cyclins determines when MPF is active.

∑ Three G₁ cyclins bind to Cdc28 to form S-phase promoting factor (SPF); SPF inactivates APC, activates expression of genes for DNA synthesis and activates cyclins 5 and 6 used to promote mitosis.

∑ Cyclins 5 and 6 are initially inhibited by the S phase inhibitor Sic1; activation of these S phase cyclins (13-25).

∑ Budding yeast cyclins -There are 8 cyclins in budding yeast; these cyclins are expressed at different phases of the cell cycle (13-26); function of different cyclin groups.

∑ Control of DNA replication during S phase (START); origin recognition complex (ORC) binds to Oriπs and forms a pre-replication complex; Cdc28-Clb complexes activate replication and inhibit new pre-replication complexes, thus only one replication (13-27).

Mammalian cell cycle control:

∑ Cell proliferation - Growth factors induce proliferation by stimulating progression through G₁ ; the cell cycle restriction point is START.

∑ There are multiple cyclins (A, B, D, E) and multiple Cdks (Cdk1, Cdk2, Cdk4, Cdk6); different combinations of Cdks and cyclins mediate progression through the cell cycle (13-29); growth factors stimulate early and delayed (cyclins D and E and Cdk 2, 4 and 6) response genes (13-30) required for passing the restriction point by regulating Rb and E2F activity (13-31); Cdk2-cyclin A is required for DNA synthesis; Cdk1 is active in G₂ phase and induces entry into mitosis (i.e. mammalian MPF).

Cell cycle checkpoints:

∑ Checkpoints afford protection for the cell during division; stages when checkpoints occur (13-34).

∑ DNA damage dependent arrest is controlled by the p53 transcription factor and p21 Cdk inhibitor (13-36).