L16 cyclins of cell cycle (CC2)

• the checkpoints of the cell cycle are the G1 checkpoint (checking to see if environment is favorable), G2 checkpoint (see if DNA has been replicated) and metaphase checkpoint (see if all chromosomes are attached to the spindle).
• checkpoints allow the cell to know what phase its in to stop. they are not needed to complete the cell cycle, but when removed or deleted, it will lead to mutations or cell death due to mutations
• cyclin-dependent kinases are the main proteins that make the cell cycle possible. in yeast there is only one Cdk, in mammallian cells there are many. they work with cyclins to make the cell cycle work. the cyclins are specific to each phase in the cell cycle.
• the different cyclin-cdk complexes that exist are: G1-cdk, G1/S-cdk, S-cdk, M-cdk
• cdk associates with different cyclins to trigger different events of the cell cycle. the binding of M cyclin with cdk triggers mitosis machinery. the binding of S cyclin with cdk triggers DNA replication machinery via phosphorylation of substrates responsible for DNA replication.
• the cycle for the creation and degradation of cyclins is cyclin synthesis -> activity (usually phosphorylation of substrates to trigger activity that's supposed to happen in that phase -> degradation of cyclins (via proteolysis)
• cyclin activates the cdk via:

  1. the T-loop is initially blocking the active site of cdk
  2. binding of cyclin causes the T-loop to move out of active site, leading to partial activation
  3. Cdk activating kinase (CAK) phosphorylates the active site of cdk to make the cdk/cyclin complex active
• cyclin/cdk is deactivated via

  option 1: Wee1 kinase that puts an inhibitory phosphate. cdc25 phosphatase can take this inhibitory phosphate away though.

  option 2: cyclin-cdk complex inhibitor proteins (CKI), p27 is an example of this where it will bind to the cyclin/cdk complex and alter the conformation and active site, leading to an inhibition of cyclin/cdk
• these cyclin-cdk complex inhibitor proteins need to be regulated to ensure that the cell does not overproduce them. this regulation is done by an active ubiqituin E3 ligase called SCF (it is substrate specific and can only recognize CKI when it is phosphorylated).

  1. a kinase phosphorylates the CKI protein
  2. SCF adds on a polyubiquitin chain with the help of an E1 and E2 ubiquitinylation enzymes
  3. this chain serves as a signal for the proteasome to degrade the CKI
• a way to regulate the degradation of M cyclin via the proteasome is through another E3 ligase, the anaphase promoting complex (APC).

  1. APC must bind to cdc20 to make it active
  2. this active APC will work with E1 and E2 to add a polyubiquitin chain to the m-cyclin
  3. this serves as a signal to proteasome to degrade the m-cyclin
• An experiment was done where cells in S phase, G1, G2 had a nucleus of another cell inserted into that cell.

  If a nucleus of G1 or G2 cell was put into an S phase cell, the G1 or G2 nucleus would go into S phase.

  If a nucleus of G2 cell was put into a G1 cell, the G2 cell would reach S phase at its own time

  This experiment illustrated that there the cytoplasmic factors for DNA replication present in the S phase cell disappear when cell moves into G2.
• system that promotes cells into S phase:

  1. DURING G1: Cdc6 binds to the ORC (origin recognition complex) on DNA. Mcm proteins will assemble into a ring of helicases to create the pre-replicative complex.
  2. TRIGGER INTO S PHASE: S-cdk phosphorylates Cdc6 (which is degraded) and ORC is then phosphorylated, triggering the creation of a replication fork (NOW IN S PHASE)
  3. DURING G2/M: ORC stays phosphorylated, so none of the proteins can allow this area to start replication over again, preventing the cell from returning into S phase
• system that promotes cells into m phase:

  1. m-cyclin will bind to cdk1 creating an inactive M-cdk
  2. cdk-activating kinase (CAK) adds an activating phosphate while Wee1 (cdk-inhibitory kinase) adds an inhibitory phosphate
  3. cdc25 removes the inhibitory phosphate, leading to an active cdc25 and an active M-Cdk
• the system that promotes cells into m phase via m-cdk results in a positive feedback loop because active M-cdk stimulates cdc25 (which activates M-cdk). M-cdk also inhibits Wee1 (which allows for activation of M-cdk)
• after metaphase-anaphase transition, there is no cdk activity, which halts the cell cycle. this is good because we don't want the whole process to start again, because if it repeats to many times you will get too many chromosomes in one cell.
• at G0, the cells do thier normal functions. they have inactive cell cycle machinery.
• the signal for G1 to start again in yeast is glucose and the signal for mammalian cells are called oncogenes (genes that push cells foward to make new cells).
• tumor supressor genes are those that stop the mammilian cell from continuing in the cell cycle
• Checkpoints primarily operate through negative signals, halting progression until specific conditions like unreplicated DNA and unattached chromosomes are met
• Cdk activity is regulated by inhibitory phosphorylation and binding of Cdk inhibitor proteins (CKIs), with Wee1 kinase inhibiting Cdk activity through phosphorylation
• Cyclin destruction by ubiquitin-dependent proteolysis, regulated by enzyme complexes SCF and APC, is a key mechanism in the cell-cycle control system
• Transcriptional regulation adds an additional layer of control in more complex cell cycles, involving the regulation of cyclin levels through changes in gene transcription and synthesis
• Insights from fusion experiments show that G1 cells are competent to initiate DNA replication, while cells in G2 phase cannot rereplicate DNA even with S-Cdk activity
• Origins of Replication Complex (ORC) is a multiprotein complex that binds to replication origins throughout the cell cycle, acting as landing pads for regulatory proteins
• Cdc6, a regulatory protein, transiently increases in early G1 phase and binds to ORC at replication origins, facilitating the formation of the pre-replicative complex (pre-RC)
• Assembly of the pre-RC marks the initiation of the DNA replication process, serving as a platform for subsequent recruitment of additional factors necessary for replication initiation
• In G1 phase, pre-RC is assembled at replication origins, and activation of S-Cdk in late G1 phase initiates DNA replication by triggering origin firing
• 1. Cdk prevents rereplication by causing the disassembly of pre-RC post-origin firing and by phosphorylating Cdc6 to prevent reassembly at any origin
• During G2 and mitosis, sustained activity of S-Cdk prevents rereplication post-S phase
• The nucleus ensures the Mcm complex cannot bind to the replication origin, preventing rereplication
• Sustained activity of S-Cdk remains high during G2 and early mitosis, preventing rereplication post-S phase
• 1. Cdk phosphorylates Cdc6 and Mcm proteins during mitosis, further preventing rereplication
• Dephosphorylation allows for pre-RC assembly, preparing chromosomes for the next round of replication
• Cyclin-Cdk complexes play pivotal roles in regulating DNA replication initiation and preventing rereplication
• Completion of DNA replication in G2 phase leaves the cell with accurately duplicated chromosomes
• Mitosis involves the distribution of duplicated chromosomes and other cell contents to daughter cells
• Events of mitosis are triggered by M-Cdk, activated after completion of S phase
• Activation of protein phosphatase Cdc25 in late G2 removes inhibitory phosphates from M-Cdk, promoting M-Cdk activation
• Tight regulation of M-Cdk activation ensures orderly progression into mitosis
• Cells with defective checkpoint mechanisms enter mitosis despite incomplete DNA replication