Bio 285 SI Notes Quiz 4Cell cycle control system:- CDK: enzyme called Cyclin-dependent kinaseo Cyclin is a binding partnero “master cell cycle regulator”o Any point in cell cycle that cells decide they need to stop  inhibit CDK- CDK is regulated in several ways:o Cyclin regulation  different cyclins for different phases of the cell cycle G1 cyclins telling CDK to target G1 proteins S phase cyclins M phase cyclinso Phosphorylation & dephosphorylation by kinases and phosphatases (1)o CDK inhibitors (CDKIs) (2)- Another way of regulation is the regulation of Cyclin levels (3)o Regulate the levels of the cyclin that binds CDK- Cyclin levels rise and fall throughout cell cycle via:o Transcription & translationo Degradation through ubiquitin-mediated proteolysis- Difference between CDK and cyclin: CDK is the car while cyclin is the keyCell cycle checkpoints:- G1 phaseo Check for DNA damage (before replication begins)o Check for favorable environmento Must have enough material to create a duplication of the cell- S phaseo Check for errors, incompletely duplicated DNA- G2 phaseo Damaged or incorrectly replicated DNAo Enough resources to continue- M phaseo Proper chromosome and microtubule attachment and arrangement- If DNA gets messed up, can stop it during those checkpoints- S & G2 do basically the same thing- Summarize whole cell cycle by putting each step in each stageOverview of cell cycle:- G1 phase  growth “gap” phaseo Could duplicate organelleso Cell checks environment Growth factor signals present? Enough nutrients? Enough energy? DNA  check for damageo Restriction point/START/G1-checkpointo Prepare for DNA replication (“prepare ingredients) Make nucleotides Make proteins required for replicationo Once replication starts cell now in S phase- S phase  “synthesis” phaseo DNA replication- G2 phase  2nd growth phaseo Check for DNA damageo Finish replicationo Prepare for M phase- M phase  chromosome segregationo Cytokinesis: splitting of the cytosol- G0 phase  non-dividing stateo Stop replicationo Cell damaged, etc.Control of the replicative cycle:- Goal is to set up where the replication begins- Replication begins at origin of recognition site on DNAo Multiple ORCs that occur at same time- Form the pre-RCo How? Get ORC to bind to origin of replication, ORC then recruits Cdc6 which recruits CDT1 which recruits Mcm, then Mcm/Cdc6/CDT1/ORC complex makes up pre-RC complex Still in G1 to S phase transitiono Once pre-RC complex together, start replication Now in S phase Have more than one ORC not all fire at one timeo Mcm is the helicase; only protein from pre-RC physically involved in replicationo Cdc6, CDT1, ORC all ATPases- Stop replication from happening a second time? Phosphorylate ORC it can’t call/recruit Cdc6 or CDT1 again, phosphorylate Mcm it leaves nucleus, phosphorylate Cdc6 it gets degradedo S-phase cyclin/CDK phosphorylates all theseo Have to do all three to stop replicationo Do all three to have multiple areas so you have more control over it Three locks on a door, low chance of them all breaking compared to having one lock and having it breakG2 phase: prepping for chromosome segregation- M-cyclin levels rise on a graph, solid line at bottom CDK activity, CDK activity quickly rises then immediately falls on graph; as M-cyclin levels drop so does CDKo S-phase: M-cyclin levels are rising but no CDK activityo M-phase: see a spike in CDK activity- CDK bound to M-cyclino M-CDK inactive at this state- 2 kinases (Wee1 & Cak) add phosphates to M-CDKo Wee1 adds a phosphate that inhibits M-CDKo Cak adds a P that activates M-CDK Have both, M-CDK still inactive even though 2 phosphates- Cdc25 takes off a phosphate from M-CDK (takes phosphate from Wee1)o Cdc25 is a phosphatase because it takes off a phosphate that takes off the inhibiting phosphate- M-CDK now active (only has phosphate from Cak)- M-CDK finds an inactive Cdc25 & activates it by adding a Po Active Cdc25 now finds more inactive M-CDKs and activates them, process then goes on and on- This is a positive feedback loop, what causes jump in graph- Why does cell do this? When moving from G2 to M phase, G2 is a growing phase, cell doesn’t want to divide too quickly, why needs inhibitory phosphate, when cell has grown enough you want it to divide so instead of having to wait for phosphate to be taken off AND cyclins to be made, easy to just have to take phosphates off & have already made cyclins- Drop in CDK activity on graph is because cyclins get degradedo Once cyclins get destroyed, CDK activity decreases- If you phosphorylate Wee1, you inhibit it (inhibiting inhibitory kinase), which increases cyclin creation (?)o Same as when phosphorylate Bad, you inhibit Bad from doing its jobSequential phosphorylation:- Final goal to move cell cycle forward & in order- Example: Have a substrate, CDK adds a P to that substrate, point here to activate PLK1, as a result PLK1 isstill inactive when it binds to the substrate its phosphorylation then its activeSpindle assembly:- Spindles made of microtubules which are made of tubulins- Tubulins are GTPaseso Made up of alpha & beta tubulin- Centrosome vs. centrioles vs. centromereso Centrosome made of centrioles  Located on spindle poleso Centromere in middle of chromosome; where kinetochore sits Segment of chromosome/DNA where kinetochores are on- **Insert spindle assembly picture- 3 types of microtubules: aster (attached to side of cell), interpolar (in middle), kinetochore (attached to kinetochore)- Microtubules have polarity – not charged, just positive or negativeo Growth happens on positive end (polymerization & depolymerization); positive end located opposite from the spindle poleo Negative polarity embedded in the spindle pole- Microtubule picture: (1) is interpolar, (2) is kinetochore, (3) is aster- Dynein walks towards negative end, kinesin walks towards positive end **- Dynein stationary, pulling pole away from it, negative end going towards dynein & positive end going away from dynein- Eg5 holds onto 2 microtubules at the same time (feet on both ends), stuck in place not moving only legs aremoving; drags microtubules to the opposite way it’s walkingo Eg5 moves towards positive endo As you push microtubule, tubulins get added to plus end so Eg5 has more length to walk on; causes poles to move away from each othero Eg5 keeps microtubules at a common distance, don’t overlap too mucho Eg5 only