Cell Cycle

• G1 - cell growth, increase in phase/size; centrosome replication in late G1 phase
• S - DNA rep
• G2 - DNA exist as chromatid, centrosome separation
• Prophase - centrosome migration; choromsome condensation; appear as long threads
• Prometaphase - spindle formation; nuclear envelope fragmentation; chromsome condensation completed and held together at centromeres by spindle microtubules
• Metaphase - chromosome alignment
• Anaphase - chromosome separation
• Telophase/cytokinesis - nuclear membranes reformation; chromsome decondensation; spindle diassembly
• Cytokinesis - cyto plasmic division by the contractile ring

mitotic cyclin (cyclin B) + mitotic cdk (cdk1 = cdc2)

• cdk1 - cyclin dependent kinase, also
• cdc2 - cell division cycle
• ser/thr protein kinase
• no acvitivy without cyclin

• Cyclin association/destruction
• Activating phosphorylation events on cdk
• Inhibitory phosphorylation events on cdk
• Cdk inhibitors (CKIs)

• a phosphatase that removes phosphate from cdh1, activating ch1 so it binds to APC/C and activate APC/C
• only actie in late anaphase

• APC/C binds cdc 20 to ub securin and activate separase to separate sister chromatids
• G1 cyclin phosphorylates cdh1, deactivates it, cdh1 cannot bind APC/C, APC/C remains inactive
• cdc14 phosphatase (only active in late anaphase) dephosphorylates cdh1 and ch1 binds to APC/C - activating it
• cdc20 and APC interaction is before cdh1; cdc20 is inhibited until all chromosomes are attached and aligned
• cdh1 activity is inhibited until the sister chromatids are separate
• functions as a checkpoint to ensure that chromosomes remain condensed and nuclear envelope does not assemble until chromosomes are properly agined

• excess of wee1 leads to elongated cells, incrased G2
• deicit of Wee1 leads to small cells, decreased g2
• wee 1 phosphorylates inactive MPF on Y15
• leads to CAK to phosphorylate (CDK activating kinase) on T161
• cdc25 dephosphorylates on Y15 will activate cdc2 (cdk1) makes active MPF

• in the budding yeast
• activates CDK, final step to make active CDK, dephosphorylate at Y15

high activity: has Thr160 phosphorylated - substrate binding site

• Nuclear lamins
• phosphorylation of Lamin tetramers by MPF initiates disassembly
• Proteins invovled in chromosome condensation, spindle assebmly, chromosome alignment

• Interphase - MPF leads to phosphorylation of lamins - disassociation
• Prophase - nuclear envelope fragment, after prophase Lamins are dephosphorylated
• Early Telophase - by then, lamins are dephosphorylated and reassembbly
• Late Telophase - by then, fusion of nuclear envelope fragments
• After telophase - fusion of enveloped chromosomes

mediates homotypic vesicle fusion

• smc2, smc4 + kleisin
• SMC protines: sturcutral maintenance of chromosome proteins
• Function: link daughter chromosomes together
• From G2(after replication of chromatin) to during metaphase, cohesion concentrates around centromere, concentration regulated by phosphorylation

• bidngs to APC/C, acivating it, so that it polyubiquitinates securin for degradation
• degradation of securin leads to release of spearase from separase + securin complex, separase is activated and cleaves kleisin (cohesion cleaved)
• sister chromatids separate

• 9 amino aacids, directs polyubiquitination on lysine, target for destructino by proteasome.
• Entry into mitosis requires accumulation of cyclin B
• Exit from mitosis requires degradation of cyclin B

• the only cdk in budding yeast cells - a cell division kinase
• cdc28=cdc2 in fission yeast = cdk1 in mammals

• s-phase promotin factor
• Budding Yeast: cyclin 1, 2 (G1 cyclin) + CDK (cdc28)

• cyclin 1 and cyclin 2 (cln1, cln2)
• clb1, clb2
• cyclin B

• S-phase inhibitor in budding yeast
• binds to SPF and inhibits

• cyclin association/destruction
• activating phosphorylation events on cdk
• inhibitory phosphorylation events on cdk
• cdk inhibitors (CKIs)
• Note: cdk protein levels remain relatively constant throughout the cell cycle

• G1/S trasition
• sic1 binds to Sphase cyclin (clb 5,6) and cdc28(cdk) and inhibits SPF (Sphase cyclin + cdc8)
• G1 cyclin (cln1) and cdc28 (cdk) phosphorylate sic 1
• leads to polyubiquitination of phosphorylated sic 1 via SCF (Ub E3 ligase) and proteasomal degradation
• required for G1/S transition - lead to DNA replication

• Skp1/Cul1/F-box protein = E3 Ub Ligase
• mediates G1 cyclin regulators degradation and activates G1 cdk, required for G1/S transition

origin recognition complex, bind to origins of replication

DNA helicase loading factor, increased in early G1, bind to Oc

DNA helicase

• pre replication complex
• ORC + cdc6 + MDM

• ORCs bind to origins of replication
• Cdc6 (DNA helicase loading factor) level is increased during early G1 and binds to ORC
• ORC, cdc6, and Mcm (DNA helicase) form a complex called pre-RC (replication complex)
• accumulated s-cdk phosphorylates cdc6
• cdc45 binds to Mcm - activates Mcm, unwinding of parental DNA strand leads to release of cdc6-p and ctd1-p
• release of cdc 6-p lead to degradation, allows: Mcm to move and assembly of replication fork (RPA, DNA polymerase)
• cdc6 is inhibited by phosphorylation, degradation and nuclear export until M-cdk is inactivated at the end of mitosis

• BrdU is a synthetic nucleoside that is an analogue of thymidine - can be incorporated into DNA during replication
• G0 cells were treated with growth factor and either injected with antibody or anti-cyclin D antibody, then added brdU, wait 16 hours
• Injection of control antibody: BrdU positive cells - rep occured, BrdU incorporated into DNA
• Injection of anti-cyclin D antibody: Cylin D bound to antibody, acnnot get in S phase, no DNA rep, BrdU-negative cells (not incorporated into DNA)
• Conclusion: Cyclin D is important for DNA replication

• Retinoblastoma protein - a tmor suppressor gene (inactivated in most human cancer cells)
• Mid G1: Rb binds E2F, inhibiting E2F
• Mid G1 cyclinD + Cdk4/6 (induced by growthfactor) and Latae G1 Cyclin E + Cdk2 both phosphorylate Rb
• Rb-p releases E2F, E2F is activiated - lead to positive feedback, further phosphorylation by S-cdks
• S cdks accumulate activated E2F lead to DNA rep and G1/S transition

• Detects damage to DNA throughout the cell cycle
• a tumor suppressor and trasncription factor, stabilized p53 activates transcription fo p21 CIP (a CDK inhibitor)
• p21 gene is translated into CDK inhibitor protein
• Mdm2 is aubiquitin ligase - p53 negatively regulated by mdm 3

• kinetochore-mediated: cdc20
• MTOC-mediated: mad2
• ensures all cell cycle is only one directional

• mad 1 binds to a kinetochore that is not connected to microtubule
• mad 2 can bind to mad 1, from open conformation to closed conformation
• another mad 2 can bind the mad1/close mad2 and become closed
• the newly closed mad 2 binds cdc20, inhibiting cdc, more open mad 2 bind closed mad2/cdc20, and bind other cdc20 to inhibit them
• inactiviation of cdc20 inhibits cdc20-APC interaction, so secruin is not degraded
• spingle-assembly checkpoint'
• cell cycel is inhibited until all the chromosomes are aligned and all kinetochores were attached by MT, mad1/2 will deatch, and bind p31

• when the MT binds to kinetochores, mad1/close mad2 tetramers are released from kinetochore and p31 binds
• p31 also binds closed form of mad 2 and frees cdc20, closed mad2 becomes opened mad2, and free p31 floating around
• cdc20 will bind APC/C
• lead to secruin degradation
• entry into anaphase

• SPB: spindle polary body, first in the mother, entrance into the bud trigger TEM1-GDP to be activated, Cdc14 was originally stored in the nucelolus, will be distributed, to that stored (inactive) cdc14 becomes active (spread out)
• TEM1-GTP is the active conformation
• if SPB remains in mother cell, TEM remains inactivated (GDP bound), cdc14 remains in nucleus, leads to mitotic arrest
• TEM1-GEF only in bud; Kin4 (TEM-GAP activator in cortext of mother cell)
• Note: cdc14 phosphatase activity is required for exit from mitosis

small GTPase that activates MAPK pathway, important for proper chromosome segregation/budding