2Outline of Class 1. History and fundamentals 2. MPF and the discovery of CDKs3. CDK regulation of mitotic entry and exit4. Regulation of G1-S5. DNA replication control6. Cell cycle checkpoints7. Discussion of papers
3Key points 1. Essential components of the cell cycle and what they do 2. Logic of cell cycle circuitry and switches3. Experimental approachesMany topics we have no time to cover, or will cover in little detail:chromosome dynamics, cytokinesis, centriole replication, cancer cell cycles,developmental/alternative cell cycles, checkpoints, DNA replication mechanisms,meiotic cell cycle, growth control
4What is a Cell Cycle? Process by which cells replicate themselves 1830’s Schleiden & Schwannorganisms are made of cells1850’s Schultzecytosol (protoplasm) and nucleus defined as separate entities in animal and plant cells1850’s Remak and Virchowomnis cellula e cellula --all cells come from other cells1880’s Fleming and Strasburgermitosis (chromosomes look like threads)1950’s Stages of cell cycle are definedDNA replication as a discrete event (S phase)1960’s Continuous and discontinuous eventsRNA synthesis, protein synthesis, cell growth--continuousDNA synthesis, Mitosis--discontinuous
5Cell Cycle Fundamentals 1 G1SG2MitosisInterphase12G1MG2SRelative AmounttimeProteinRNADNAMass/SizeRestrictionPointS phase and Mitosis are defined by processesG1 and G2 (gap phases) are defined by timingG1 (and G2?) can be split into more meaningful sub-stages by molecular and physiological criteria:Restriction Point in mammalian tissue culture cellsdefined by serum sensitivity
6Cell Cycle Fundamentals 2 Mitosis is subdivided into different stagesMitosis involves the formation of a mitotic spindle with all the chromosomes properly aligned (A-D),followed by mitotic exit where sister chromosomes are separated, a cleavage furrow forms, nuclear envelope re-forms, and chromosomes decondense.
7Cell Cycle Fundamentals 3 The cell cycle can be broken into subcycles whose relationships change in different cell types and under different circumstancesTiming in a typical somatic cell:1. DNA replicationS phase2. Centrosome duplication(microtubule organizing center)S phase3. Nuclear Division (karyokinesis)Mitosis4. Cell Division (cytokinesis)Mitosis5. Cell GrowthThroughoutIn a typical somatic cell 1-5 are all regulated and coupled
8Cell Cycle Fundamentals 4 Non-canonical cell cycles are found throughout nature and play a criticalrole in cell and developmental biologySomatic cell 1-5 regulated1. DNA replication3. Nuclear Division (karyokinesis)4. Cell Division (cytokinesis)5. Cell Growth2. Centrosome duplication(microtubule organizing center)Embryonic cell cycle: 1,2,3,4 (division with no growth)Meiotic cell cycle 2,3,4: (division with no DNA replication)Megakaryocytes, slime molds:1,2,3,5 (replication and nuclear division)Liver cells, fly salivary glands, many plant tissues:1,2,5 (endoreplication=S phase with no mitosis)Oocyte formation: 5 (growth with no division)Some green algae: 5 and 1,2,3,4 separated (Chlamydomonas)or 1,2,3,5 and 4 separated (Scenedesmus)Ciliated epithelial cells, some protozoans:2 (centriole amplification)
9Cell Cycle Fundamentals 5 Cell Cycle States are RegulatedI. Johnson and Rao (1975) Cell fusion experiments
10Cell Cycle Fundamentals 5 Cell Cycle States are Regulated:nucleocytoplasmic ratio controls replicationII. (Sudbery and Grant, 1976) Physarum (slime mold)Starved plasmodiumno growth,no DNA replicationUV irradiate~50% of nuclei inactivatedRemaining nuclei replicate and divide untilnucleo-cytoplasmic ratio is restored
11Cell Cycle Fundamentals 6 Models for Cell Cycle AnalysisXenopus oocytes/extractsSimple biochemical systembudding yeast (Saccharomyces cerevisiae)Powerful genetics, G1-S regulationfission yeast (Schizosaccharomyces pombe)Powerful genetics, G2-M regulationmammalian tissue culture cellse.g. HeLa cells, NIH3T3Closest model for human cells, regulation is more complexDetails vary between organisms, general principles are similar
12Xenopus oocytes/extracts Properties of Xenopus system:Cell free extracts can cycle betweenS phase and MitosisExtracts can be manipulated to effect amitotic arrest: chelate calcium > stabilizecytostatic factor (CSF)Feedback controls/checkpoints are missing*-extracts can cycle without nucleiG1 regulation is absentMost components needed to drive cell cycle are already present in the cytoplasm to support rapid embryonic cell cyclesNo transcription necessary. Only a single protein needs to be translated to drive the cell cycle forward.If enough nuclei are added then feedback regulation and G1 intervals appear.
13Discovery of MPF (maturation promoting factor) Serial injections can be repeatedindefinitely until original sourceof MPF is diluted awayYoshio Masuiand co-workersMPF activity fluctuates and is presentin both oocytes and fertilized embryosTim Hunt andco-workersCyclin synthesis and abrubtdegradation mirrorsrise and fall of MPF activity
14Biochemical nature of MPF I Lohka andMallerStabilize MPF in vitro from Xenopus extracts and fractionate:MPF has two components 32 kd and 45 kd; MPF posseses kinaseactivity45 kd protein = cyclin BKinase activity only present when both subunits are presentSeveral groups:Murray andKirschnerTake cell free extract and RNase treatto destroy all endogenous mRNAsInactivate RNase with inhibitor and add no RNA or cyclin mRNAactivate extract to induce interphase and add sperm nucleiPart IMPF appears to have a kinase activity--is this rigorous proof? Could it be other proteins in the prep?Histone H1 is a good in vitro substrate for CDKs, often used to assay CDK activity
15Biochemical nature of MPF II Murray andKirschner∆13 equiv. to wt cyclin B∆90=nondegradable cyclinmissing destruction boxCyclin synthesis drivesactivation of MPFCyclin destruction is requiredto inactivate MPF and drivecells into interphaseMPF is required forcyclin destructionOscillator behavior of cell cycleis explained:cyclinMPFPart I--what does panel b show, what about panel c?PartII-what is paradoxical about this result?Part III-In lower panel the cells enter anaphase but the chromosomes remain condensed, suggesting high mitotic activity. What does this suggest about regulation of anaphase?Cyclin destruction is NOTrequired to initiate anaphase
16Yeast Genetics and Unification of the Cell Cycle Main control point is G2/MboundaryMain control point is G1-S boundary
17cdc mutants are critical for identifying cell cycle components Hartwell and colleagues, Nurse and colleaguesbudding yeast fission yeastG1-S blocked cdc mutantWhy did Hartwell and Nurse look for temperature sensitive mutants?Would you expect most ts lethal mutants to have a cdc block?cdc28 (budding yeast) has two arrest pointspre-S phase, and pre-Mcdc2 (fission yeast) shows arrest at G2/M, butalso has dominant alleles that give a weephenotypephenotypes suggest that these two cdcs havea critical role in cell cycle regulationmitotic cdc mutant
18Unifying observations for the cell cycle field budding yeast CDC28 = fission yeast cdc2 = Xenopus MPF 32kd subunit aka CDK1budding yeast daf1/whi1 = a cyclin (later renamed Cln3)fission yeast cdc13 = a B-type cyclin homologKey enzyme for cell cycle regulation is now defined as acyclin dependent kinase complex (CDK) composed of acatalytic kinase subunit and a cyclin that activates the kinaseSince then things got more complicated: Multiple CDKs, Multiple cyclins andInteracting proteins discoveredAll eukaryotes use the same set of proteins for cell cycle regulation withsome species specific variation2001 Nobel Prize in Physiology or Medicine given toTim Hunt, Lee Hartwell, and Paul Nurse for their pioneering work incell cycle regulation
19Understanding the somatic cell cycle Somatic cells and yeasts have a G1 period with low CDK activitySomatic cells and yeasts have multiple cyclins or CDKs that controlprogression through G1, S phase and mitosisSomatic cells and yeasts have feedback controls that gate eachtransition to ensure proper completion of previous events
20Nomenclature How to deal with all these gene names Proteinyeasts buddingfissionanimalsplantsG0/G1 CDKsCdc28cdc2CDK3 (G0), CDK4, CDK6CDKAS CDKsCDK2, CDK1CKDAG2/M CDKsCDK1CDKA, CDKBG0/G1 cyclinsCln1, Cln2, Cln3puc1Cyclin D, Cyclin C (G0)D cyclinsS cyclinsClb,5,6cig2Cyclin E, Cyclin AA cyclinsG2/M cyclinsClb 1,2,3,4cdc13Cyclin BB cyclinsWhy might cyclin and CDK gene families become expanded? What regulatory possibilities does duplication allow?
21CDK Regulation How is CDK activity controlled during the cell cycle? Activation by cyclin bindingActivation by phosphorylation (CAK)Activation by dephosphorylation (CDC25, Cdi1)Activation by destruction of inhibitor (Skp1-Cullin-F box complex aka SCF)Inactivation by cyclin destruction (Anaphase PromotingComplex/Cyclosome aka APC/C)Inactivation by phosphorylation (Wee1)Inactivation by inhibitory binding proteins (KIP/CIP/WAF/KRP and INK)Substrate specificity (CDK-cyclin combinations)Subcellular localizationCDK abundance usually not regulated
22CDK regulation I Why is CDK kinase activity non-linear with respect to cyclin concentration?Cyclin concentrationor CDK activitytime
23CDK activation and inactivation by feedback loops wee1Inhibitorykinasecdc25ActivatingphosphataseCAK (CDKActivating Kinase)CKI/KIPICK/KRPCdc2-cyclin BCell cycle targetproteinsUbiquitinligase for cyclinAPC/C
24Structural basis for CDK activation by cyclins and phosphorylation I Inactive monomerATP misoriented, substratebinding occluded by T-loop,PSTAIRE helix mispositionedCyclin boundATP properly oriented via interaction with repositioned T loop and PSTAIRE helix.Substrate binding cleft suboptimal.Tyr14 site in roof of ATP binding cleftis available for Wee1phosphorylation(not shown)
25Structural basis for CDK activation by cyclins and phosphorylation II CDK Thr160 +cyclinT loop flattened. Phospho T160forms stabilizing interactions thatoptimize binding siteWith substrate peptideSPXK-containing peptidefits into pocket and interactswith T loop,including Phospho T160.
26Phosphorylation/Dephosphorylation of CDKs cdc2CAK (CDK activating kinase) --largely unregulatedWee1 Major negative regulatorCdc25 Major positive regulatorBalance between Cdc25 and Wee1 activitiesregulates mitotic entryPredict the phenotype o:f a cdc25 overexpression mutant; of a cdc2 Y15F mutation; a cdc2 Y15D or Y15E mutation.
27Postive Feedback Loop for CDK Activation inactiveactiveWe won’t talk about much about mitotic CDK substrates, but they are obviously very important.What criteria would you use to establish that a protein is a substrate for a mitotic CDK?-phosphorylated in mitosis-can be phosphorylated by highly purified or recombinant mitotic CDK in vitro on same sites as in vivo-Loss of in vivo phosphorylation when kinase is inhibited or knocked downVery hard to prove conclusivelyLoop leads to explosive auto-activation of CDK once its activity rises above a certain thresholdWhat are the substrates CDK1-cyclin B that lead to mitotic entry and progression?Cdc25, Histone H1, lamins, cyclin B and many more
28Mitotic Exit is Regulated APC/C I APC/C is a E3 specificity factor for ubiquitin ligase pathwayexamples:B cyclinCdc20Cdc20++orPds1orCin8Hct1activator andspecificity factorRXXL destruction (D) box-containing substratesCdc20, Hct1 (Cdh1)KEN-box substratesHct1 onlycore complexKENRXXLHow would you show that a destruction box is necessary and sufficient to target a protein for degradation?-delete and stabilize protein-move to heterologous protein and confer mitotic instabilityCdc20 was isolated as a cdc mutant. What is its arrest phenotype?Targeting by APC/C leads to rapid degradation by the 20S proteasome
29Mitotic Exit is Regulated APC/C II B Cyclins and Pds1(Securin) are key substrates of APC/C:Nondegradable cyclin blocks MPF destructionbut does not block anaphaseAPC/C has at least one more target whosedestruction promotes anaphasePds1/Securin destruction releases a protease,separase, that degrades cohesisns andallows sister chromatids to separate
30Budding yeast mitotic exit Decreased CDK activity and Separase release activate FEAR(Cdc14 early anaphased release)Cdc14 is a protein phosphatase that plays a central rolein exiting mitosisCdc14 dephosphorylates and activates cdh1 subunit of APC and Sic1(a CDK inhibitory protein) to establish a stable G1 state with low CDK activityCdc14 activates a second pathway called MEN (mitotic exit network) that initiates cytokinesis
31Overview of APC activation and mitotic exit CDK1-Cyclin B activates APC-Cdc20 directly or indirectly through phosphorylationA time lag between APC-Cdc20 activity and other essential mitotic events is essentialDecreased CDK activity allows activation of Cdc14 mitotic exit pathway, Cdh1/Hct1 andestablishment of a stable G1 stateRegulation of APC/C by CDK1-Cyclin B generates a negative feedback to drive mitotic exit
32G1 and G1-S regulationG1 is characterized by low CDK activity and high APC-Cdh1 activityWhat triggers initiation of S phase and cell cycle re-entry?
33G1 and G1-S regulation IG1 is a major control point for most cell types:Growth factors present and extracellular conditions favorable: S phaseMG1Differentiation factors present, unfavorable conditions: G0(temporary or permanent withdrawal from cell cycle)What triggers entry to S phase, what mechanisms prevent it?Cells must be growing and have reached a minimum sizemammalian cells must not contact neighbors (contact inhibition)APC-Cdh1 must be inactivated to allow S phase cyclin accumulationCDK inhibitory proteins must be destroyed or titrated awayIn budding yeast and animal cells G1 CDK activity must reach athreshold value to trigger S phase
34G1 control points Restriction point G1 S Serum Dependent Independent Restriction point in animal cells occurs late in G1Serum withdrawal before R point--cells arrest in G1Serum withdrawal after R point-cells complete S, G2 and MRestriction pointG1SPardee (1974)SerumDependentIndependentBudding yeast cells have a G1 control point termed StartRemove nutrients prior to Start--G1 arrestRemove nutrients after Start--cells complete S, G2 and MStart
35G1 and G1-S regulation II Similarities of Cln3 and D cyclins: animal cellsGrowth factorsin serum e.g. FGF,PDGFactivation ofRTK signalingtranscription ofD cyclinsbudding yeastnutrients (glucose, nitrogen etc.)increased proteintranslation rateIncreased translation of Cln3Similarities of Cln3 and D cyclins:messages and proteins are low abundanceproteins are highly unstablelength of G1 highly sensitive to dosage and expression levelscontrol rate limiting step in G1-S transitionneither are essential!
36Triggering Start in budding yeast I G1 cyclins Cln1, Cln2, Cln3Sic1--CDK inhibitor--disrupts CDK active site, prevents ATP bindingTranscription factors SBF, MBF--activators of Cln1, Cln2 and other S phase genesWhi5--negative regulator of SBF, MBFSCF--Skp1-Cullin-Fbox--E3 ubiquitin ligase targets G1 substrates (Elledge and Harper 1996)substrateCullinF-box protein(adaptor)Skp1Cdc4 is F box adaptor for Sic1F-box proteins are specificity factors in SCF,often require phosphorylation for binding targetIn early G1: Sic1 and APC-Hct1/Cdh1 are dephos. and active. CDK activity is low
37Triggering Start in budding yeast II Cln3-Cdc28As Cln3-Cdc28 activity builds:SBF/MBF become activeWhi5 is phosphorylated anddissociates from SBF/MBFCln1/2 and Clb5/6 are madeSBF/MBF are further activatedin a positive feed back loopSic1 and Hct1 are inactivated ina negative feedback loopWhi5SBFor MBFCln1Cln2?Clb5Clb6Cln1/2-Cdc28What would the phenotype be of a whi5 mutant? a cdc4 mutant? a Cln3 mutant?SCF-Cdc4Sic1Clb5/6-Cdc28Hct1/Cdh1
38Sic1 inactivation is key for S phase initation Tyers and colleaguesCdc4 binding of Sic1depends on6+ phosphorylationsTriple mutant ∆cln1 cln2 cln3 is inviable∆cln1 cln2 cln3 sic1 mutant--viability is rescued!Sic1 is key target of G1 cyclinsSic1 becomes multiply phosphorylated by CDKs during G1 followed byabrupt degradationReplacement with a high affinity Cdc4 binding site causes premature S phase initiation and genome instabilityCooperative phosphorylation ensures that a very high CDK activity must be present in order to degrade Sic1, and that Sic1 is degraded abruptly.
39G1 control in mammalian cells G1 cyclins D1-D3-(like Cln3)CDK4, CDK6-specific for D cyclinsG1-S cyclin E (like Cln1/2)CDK2-binds E and A cyclinsCDK inhibitors p27 Kip1 (homologous to Sic1), INK family (no homolog in yeast)E2F complexes (transcription factors for S phase genes, Cyclin E (like SBF/MBF)RB/p107/p130--E2F repressors (like Whi5)SCF-Skp2--targets free cyclin E and p27 for degradation (like SCF-Cdc4)
40Establishing functions of G1 CDKs and cyclins Can’t easily make cdc mutants with diploid mammalian cellsStrategies for genetic analysis:dominant negativesoverexpression (transfection)knockouts in ES cells, whole mice, or cell lines from KO micesiRNA-mediated knockdownsWhy might a dn allele of CDK2 cause cell cycle arrest, yet the null mutation has almost no phenotype?Are knockouts always a better way to determine gene function?-redundancy-inappropriate substitution of related proteinsHarlow and colleaguesDominant negative CDK mutationsFor many years CDK2/CycE thought to be a linchpin of G1-S regulationHowever, CDK2 and CycE KO cells have only mild S phase entry defects!
41Negative regulation of G1-S is critical for animal cells Unregulated cell division leads to defects in tissue morphogenesis, development and cancerSeveral G1 regulators are tumor suppressors or oncogenes-RB, INKs, D cyclins, CDK4Two classes of negative regulators:CDK inhibitorsINK4 (CDKN2) family specific for CDK4/6-Cyclin D complexesp21, p27,p57 proteins inhibit all CDKsRB-related proteinsRB, p107, p130--bind to E2F complexes and repress S phase transcriptionRegulators show some functional overlap and tissue specificitye.g. RB is expressed in cycling cells, p107/p130 in quiescent cells,p27 is constitutively expressed,p21 is induced by checkpoint activation,p57 is expressed in neuronal cells, p16INK4b induced by negativegrowth factor TGF beta
42INKs and KIPs inhibit CDKs in different ways cyclinConformation change inCDK blocks cyclin bindingCDKcyclinKipBinds CDK-cyclin, blocksATP binding and substrateaccessEarly G1--INKs keep CDK4/6 cyclin D inactive, p27 keeps CycE-CDK2 inactiveAs CycD accumulates it overcomes INK binding to CDK4/6CycD-CDK4/6 complexes compete p27 from CycE-CDK2 promoting S phase
43Phosphorylation of RB is a key step in S phase activation E2F-DPRBCycECycAS phase genesE2F-DPRBPRestriction point/late G1/SRB hyperphosphorylatedby CDK2-CyclinE complexesdissociation from E2F-DPE2F-DPRBPMid G1 RB partially phosphorylatedby CDK4/6-D cyclins (priming)Early G1 RB hypophosphorylatedE2F-DPRBPWhat is predicted phenotype of a RB phosphorylation site mutant?RB is phosphorylated by at least two kinase complexes. How would you figure out their relative contributions and whether the phosphorylations needed to be sequential?Loss of one copy of RB leads to tumorsAnimal DNA tumor viruses produce proteins (e.g. SV40 T antigen) that inactivate RBPlant DNA viruses have evolved the same trickCycD and CycE overexpressed in many cancers
44Does RB phosphorylation=Restriction Point? Previous work on bulk synchronized cells indicates correlation betweenRB phosphorylation, Cyclin E transcription and Restriction PointZetterberg and colleaguesTime lapse videomicroscopy on single cells + immunofluorescenceto look at timing of RB phosphorylation vs. R vs. S phaseHow do you produce synchronous cells?Yeast-pheromomone arrest, elutriation, cdc temperature arrest, HU (S phase)Animal cells-mitotic shakeoff, double thymidine block, colchicine arrest at mitosisHow do you measure S phase?FACS, BrdU incorporationWhat is the molecular correlate of the Restriction point?
45G1 control in mammalian cells CycA-CDK2 alsoblocks E2F DNA bindingby phosphorylation
46Parallel Mechanisms of G1 regulation in budding yeast and metazoans
47Coupling cell size to cell cycle progression yeasts show evidence of size controlnutritional shift experiment:move cells from rapid growth to slow growth conditions-observe a G1 (budding yeast) or G2 (fission yeast ) delay until a minmalsize is reachedHow could one show a that a size checkpoint exists for mammalian cells?sort G1 cells into different size classes and measure G1 durationprolong prior cell cycle so that larger daughters are produced, look for shortened G1Follow single cells by video microscopy, look for correlation between size and S phaseData on animal cells is controversial but evidence for G1 size control exists.Growth may also be cell cycle regulated:RB controls rRNA synthesisTumors often have aberrant growth characteristicsfaster cell cycle, faster growth
48Regulation of DNA replication I Properties of DNA replication in eukaryotes:Occurs at a specific phase of the cell cycle--SInitiates from specific locations termed origins--well defined in budding yeastpoorly defined in other organismsOccurs once and only once per S phaseCompletion of S phase is ensured by checkpointsS phase is regulated by oscillating CDK activityLow CDK activity required to prime originsHigh CDK activity required to fire origins and block re-priming
49Key Components of S phase Regulation Cdc6 and Cdt1-- origin priming proteins-activity is tightly regulatedOrc--origin recognition complex--binds origins throughout cell cycle, requiredfor origin firingMcms--(mini-chromosome maintenance)-part of a hexameric origin unwindingcomplex required for initiation, AAA ATPase familyCdc7-Dbf4--kinase complex analogous to CDK-cyclin required for origin firingGeminin (metazoans only) inhibits Cdt1 mediated MCM loading at originsS phase CDKs--Clb 5/6-Cdc28 in budding yeast, CycE-Cdk2, CycA-Cdk2 in mammals
50How to ensure one round of replication? Origin “Licensing”Blow, Laskey and coworkersNaked DNA + interphase Xenopus extractChromatin assembly, NE assembly, 1 round of DNA replicationMitosisNext round of DNA replicationAdd replicated G2 nuclei to fresh interphase extractcontrolNo replicationNext round of DNA replication+NE permeabilizingdetergent lysolecithinSomething present in early interphase extracts that allows replication: licensing factorLicensing factor cannot cross NE. Lf gets made in early interphase, destroyed during Sremade during M
51Model for Licensing Factor (A) licensing factor (+) generated during M-G1 transition(B) + binds to chromatin prior to NE assembly(C) further access to + restricted by NE(D) nuclear + destroyed (-) upon S phase initiation(E) cytoplasmic + decays during late S and G2Details vary between organismsPrinciples of model proved correct:Highly regulated and labile factors are generatedin G1 that bind to origins (Cdc6, Cdt1)Cdc6 and Cdt1 allow loading of MCM complex prior to S phaseS phase CDK activity simultaneously fires loaded origins andinactivates or destroys Cdc6, Cdt1, MCMsMultiple redundant mechanisms are involved
52Pre-RC formation (licensing) involves ordered loading of proteins Crystal structure of archealMCM complexBlue indicates+ charge regionthat couldaccommodatess or ds DNA
53Molecular View of Licensing in Metazoans Cdc6, Cdt1 and MCM complex can only load onto origins in G1After S phase CDK activation:Geminin (A APC-Cdh1 substrate) is stabilized and inhibits MCM loadingorigin unwinding by MCM triggers SCF-Skp2 mediated degradation of Cdt1phosphorylation of priming proteins blocks activity, nuclear entry or origin binding
55Cell Cycle Checkpoints Checkpoint: mechanism to ensure that the next cell cycle stage is notentered until the events of the current stage are completedExamples of important checkpoints:Spindle assembly--ensures all chromosomes attachedto spindle prior to anaphaseS-phase completion--ensures that replication is completeprior to mitotic entryDNA damage-blocks S phase initiation in G1 cells,blocks G2-M in S phase cells until DNA damage repaired
57Cell Cycle Checkpoints II What defines a checkpoint?Problem that sends a signal:--DNA damage--kinetochore unattached to mitotic spindleSignal detector and transducer:--DNA damage sensing kinase cascade--spindle attachment monitors-BUB/MAD proteinsTarget or Effector:--Cdc25 inactivated by DNA damage--G2-M block--APC Cdc20 inactivated by spindle checkpoint (Metaphase block)Checkpoints are often not essential under normal circumstancesi.e. unperturbed cell cycleHow to find them?
58Genetic screens for DNA damage checkpoint mutants Isolate mutants that are hypersensitive to DNA damaging agentsRescreen for those that don’t arrest cell cycle properlyHow could you isolate spindle assembly checkpoint mutants?
59DNA damage checkpoints Sensors--Mre11 complex?Most of these proteinsare conservedsignaltransducersp53 is a metazoanprotein thathelps decide whether adamaged cell arrests orcommits suicideTargets
60Enough Already! I will make lecture notes and references available on the class web site soon