Lecture 10G1/S Regulation and Cell Cycle CheckpointsOutline:G1/S regulation and growth controlG2 repair checkpointSpindle assembly or mitotic checkpointPaper: The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and timeG1/S is the major control point: in budding yeast in mammalian tissue culture cellscell growthnutrient availabilitysignals from other cellsregulated by:Cdk1 = CDC28 = cdc2cdc28 mutations - arrest with no budanalogous to MPF:SPF = S-phase Promoting FactorCdc28/cyclin heterodimerhow to identify cyclin partner? look for high copy suppressors of cdc28ts mutationsBudding Yeast G1/S RegulationCLN1, CLN2, CLN3 genes identifiedhomology to mitotic cyclins (“cyclin box”)one CLN gene sufficient, triple knockout lethalCln necessary and sufficient to induce passage through startSic1-CDK inhibitorInduced by Cdc14 phosphatase during exit from mitosisinhibits MPF/ -type cyclins, promtes exit from mitosisBinds and inhibits S-phase Clb-Cdc28 complexes Clb 5 and 6- S phase cyclinsno effect on G1 Cln-Cdc28 complexesSic1 degradation causes S-phase onsetrecognized by ubiquitination machinery when phosphorylated by G1 Cln-Cdc28induces exit from M-phase by inhibiting B-type cyclinspromotes one transition:Two Roles for Sic1-CDK inhibitorClb5, Clb6Cln1,2,3provides barrier to S-phase that must be overcomeinhibits another transition:Sic degradationSomething other than APC must degrade Sic1 in SSCF = Skp1-cullen-F-box-protein complexUbiquitin E3 ligase-analogous to APCSCF Structure and Targetsnot just for cell cycle regulation, also signal transductionSkp2 provides specificity for substrateWhat generates specificity?APCAccessory factors:Cdc20 kinase?:Clb3, Clb5, Pds1Cdh1 kinase?:Clb1, Clb2, Clb3SCFF-box proteins:Cdc4 kinase:Sic1Grr1 kinase:Cln1, Cln2model for progression through G1/SCdc28-Cln3 induced at proper cell sizephosphorylates and activates transcription factors, CLN1 and CLN2, and DNA replication enzymesAPC phosphorylated and inactivatedClb5 and Clb6 (S-phase cyclins)late G1 - CLB5 and CLB6 Cdc28-Clb5, Cdc28-Clb6 immediately inactivated by Sic 1active S-Cdk initiates DNA replication(Cdh1)Cdc14mitotic exit and G1 progression in budding yeast S Cyclin synthesis inducedSCFSic1 degraded by SCFM-Cdk inactivation by Sic1 and APCG1-Cdk activity increases - not susceptible to Sic1S-Cdk inactive until phosphorylation of Sic1 and Cdh1 by G1/S-CdksPossible mechanism to coordinate cell growth and cell cycle progressionCln3 synthesized in parallel with cell growthHow is threshold level reached?Cells inherit fixed amount of inhibitor (DNA?)Cell cycle control in mammalian cells“mitogens” required to stimulate proliferation = growth factors (in serum)quiescent cells in G0 phasemultiple Cdksmultiple Cyclinsmany cloned on the basis oftheir ability to complementyeast mutationsCdk4, Cdk6G1 D CyclinsG1-S Cyclin ECdk2S Cyclin ACdk2G2/M B CyclinsCdk1Phase Cdk CyclinG1/SD-type cyclin requiredDNA BrdUCyclin D antibodyinitiation of DNA synthesis blocked by antibodyWhat happens downstream of growth factors?early: Fos, Jundelayed: E2F, D Cyclins, Cyclin E, Cdk2, 4, 6transcription factors activated post-translationallyearly genes induce transcription of delayed genesRegulation of E2F transcription factorsE2Fs induce: themselves Cdk2 Cyclin E, Cyclin AE2F Inhibited by retinoblastoma protein (Rb), p107, p130Rb inhibited by phosphorylation target of Cdk4,6-Cyclin DE2F and Rb are tumor suppressorspositive feedback loopRb phosphorylation maintained until Cyclin B destructionSRequired to progress through S phaseCdk2-Cyclin ARequired for Cyclin B accumulationphosphorylates and inactivates Cdh1blocks cyclin B degradation by proteosomeE2F Cyclin A Cyclin BCdh1Control of G1 progression in mammalian cellsG1-Cdk induced by growth factor, phosphorylates RbE2F induces more of itself, and S phase Cyclins (E, A)S-Cdks further phosphorylate Rbmore S-Cdks accumulate - DNA replicationCC Regulation is VERY Complexreally a bit simpler...yeastCyclins are regulated by proteolysisbudding yeastcell cyclemany cyclins, one CDKCyclin Expression in Budding YeastCln3Cln1, Cln2G1Clb5, Clb6Clb4, Clb3SActivate replication originsClb1, Clb2MSpindle assembly,anaphaseSense cell sizeCommit to divisiondifferent Cdks and CyclinsMetazoansWhat generates Cdk/cyclin substrate specificity?Cdk subunit?Cyclin subunit?some mediate interaction with substrates Cyclin D-Rbhow are diverse events induced?only slight differences in substrate specificityboth Cdk1 and Cdk2 can rescue cdc28 mutationsdifferent affinity for inhibitorsBut: a single Cyclin can allow fission yeast to grow normallyCell Cycle Checkpointsor irradiationEarly Evidence for a DNA Damage / Defective Replication Checkpointcaffeine blocks arrest and allows abnormal entry into mitosisDNA damage causes arrest in Stissue culture cells - 1974How to identify genes involved in checkpoint function?YEASTIdentify mutants that cannot recover from DNA damage2 classes: repair deficient arrest deficientrepair deficientarrest deficientConserved elements of DNA damage and replication checkpointsI. SensorsProteins with functional analogs in DNA replicationrecognize damageload onto DNAcell cycle arrestactivate DNA repairmaintain arrest until repair completere-initiate cell cycle progressionor APOPTOSISIII. Effector output:II. Transducers: = kinasesATM and ATR Chk1 and Chk2phosphorylate substrates affecting protein activity or stability(inhibited by caffeine)DNA damage pathway in budding yeastionizing radiationRAD9MEC1 (ATR kinase)RAD53CDC5 (polo kinase)CLB/CDC28mitosisCHK1PDS1cohesinsanaphase entryG2 or M arrestExamples of pathways that block the cell cycle:Another feedback pathway in fission yeast: DNA damage results in G2 arrestTwo genes identified: chk1, rad24Both act through cdc25Cdc25Y15 ppaseDNA damage14,3,3 proteinwith NESMechanism: Sequester Cdc25 away from Cdc2Cdc25 phosphorylated by Chk1P-Cdc25 recognized by Rad24Rad24 transports P-Cdc25 out of nucleus Cdc25 cannot dephosphorylate nuclear Cdc2translocation has not been confirmed in other organismsAlso a faster response recently identified,independent of p53Cyclin E inhibition and Cyclin D degradation by APC ATM kinaseCyclin D-Cdk4,6Cyclin E-Cdk2p53 = tumor suppressor/transcription factor: stabilized in damaged cellsHigher Eukaryotes: G1 arrest mediated by p53induces expression of Cdk inhibitor p21CIPapoptosisSpindle / Mitotic CheckpointsKinetochore-mediatedsenses when all chromosomes have