Molecular Biology Lecture 2: Regulation of Cell Division PDF

Summarize the regulation of the cell cycle based on the checkpoints and their role in regulation of gene and protein activity List the steps and stages of the cell cycle Summary of Cell Cycle Regulation 1. Steps and Stages of the Cell Cycle: G1 Phase (Gap 1): The cell grows and prepares for...

Summarize the regulation of the cell cycle based on the checkpoints and their role in regulation of gene and protein activity List the steps and stages of the cell cycle Summary of Cell Cycle Regulation 1. Steps and Stages of the Cell Cycle: G1 Phase (Gap 1): The cell grows and prepares for DNA replication. It performs its normal functions and may also prepare for cell division. S Phase (Synthesis): DNA replication occurs, where the cell duplicates its chromosomes, ensuring that each daughter cell will have an identical set of chromosomes. G2 Phase (Gap 2): The cell continues to grow and prepares for mitosis. It ensures that all DNA has been replicated accurately and makes final preparations for cell division. M Phase (Mitosis): The cell divides its nucleus and cytoplasm to form two daughter cells, each with an identical set of chromosomes. Mitosis is further divided into prophase, metaphase, anaphase, and telophase. Cytokinesis: This is the final step where the cytoplasm divides, resulting in two separate daughter cells. In summary, the cell cycle is tightly regulated through checkpoints that ensure proper cell growth, DNA replication, and division. The restriction point in the G1 phase is crucial for determining whether a cell will proceed to DNA replication, and involves key regulatory molecules like cyclins and CDKs. These checkpoints help maintain genomic stability and prevent uncontrolled cell division. G0 = exit from the cell cycle This exit is NOT an elimination of metabolic activity. Cells are alive and trying to be active; however, they are no longer capable of cellular division. Once a cell has exited the cell cycle into G0, they become senescent and are unable to return to active division without further changes. These changes often involve bypassing measures in place to prevent the cell from inappropriate division. G0 can be entered from either G1 or G2. - G1 senescence is triggered by changes in chromosomes or DNA damage - G2 senescence is triggered by DNA damage only.  Match the stages with the discussed activities taking place during the stage 2. Matching Stages with Activities: G1 Phase: ○ Activities: Cellular growth, synthesis of proteins and organelles, preparation for DNA replication, and monitoring of external signals. S Phase: ○ Activities: DNA replication, chromosome duplication, and repair mechanisms to ensure accurate DNA synthesis. G2 Phase: ○ Activities: Further cell growth, production of proteins necessary for mitosis, DNA repair, and verification of proper chromosome duplication. M Phase: ○ Activities: Chromosome alignment, separation of sister chromatids, and cytokinesis. G1/S Checkpoint: Definition: The G1/S checkpoint, also known as the restriction point, is a critical regulatory point in the cell cycle that determines whether a cell is ready to enter the S phase (DNA synthesis) from the G1 phase (Gap 1). Purpose: This checkpoint ensures that the cell has adequate resources and favorable conditions to proceed with DNA replication. It checks for DNA damage, sufficient growth signals, and appropriate size and nutrient levels. G2/M Checkpoint: Definition: The G2/M checkpoint is a control mechanism that ensures the cell is ready to enter mitosis (M phase) from the G2 phase (Gap 2) of the cell cycle. Purpose: This checkpoint verifies that DNA replication is complete and that there are no DNA errors or damage before the cell undergoes mitosis. It also checks for proper chromosome alignment and readiness for mitotic spindle formation. Spindle-Assembly Checkpoint (SAC): Definition: The spindle-assembly checkpoint is a safety mechanism during mitosis that ensures all chromosomes are properly attached to the mitotic spindle before proceeding with anaphase. Purpose: This checkpoint prevents premature separation of sister chromatids until each chromosome is correctly aligned and attached to spindle microtubules, thereby ensuring accurate chromosome segregation. ○ MAD2 is a key component of the spindle assembly checkpoint (SAC), which ensures that chromosomes are properly attached to the spindle microtubules before anaphase begins. If MAD2 is constitutively activated, it will continuously inhibit the anaphase-promoting complex/cyclosome (APC/C), preventing the cell from progressing from metaphase to anaphase, thereby halting mitosis  List the steps associated with commitment to DNA replication (restriction point) and identify the molecules involved 3. Commitment to DNA Replication (Restriction Point) and Associated Events/Molecules: Restriction Point (R-Point): ○ Critical checkpoint where the cell decides whether to proceed with DNA replication and enter the S phase. ○ Series of events that must occur to enter S phase but not in a particular order Events Involved: ○ Hyperphosphorylation of Rb Enables previously stalled cells to synthesize DNA and progress Activates E2F-dependent transcription ○ Activation of E2F-dependent transcription Activated when Rb is phosphorylated, leading to the initiation of genes required for DNA replication. ○ Increased activity of CDK2 When genes are turned on, CDK2 can be upregulated and trigger activation of CDK2 Leads to further phosphorylation to progress replication 1. Explain the role of cyclins and cyclin-dependent kinases in the cell cycle with particular emphasis on the activity threshold and cyclin-CDK pair associated with each stage of the cell cycle ◦Compare and contrast cyclins and CDKs in terms of availability and activity Aspect Cyclins Cyclin Dependent Kinases (CDKs) Role in cell cycle - Regulatory proteins - Enzymes that, when that activate CDKs activated, at the appropriate phosphorylate times during the cell target proteins to cycle drive the cell cycle forward - Ensure the cell divides at the correct time Activity - Their activity is - Utilize ATP to add specific to the cell phosphate groups  cycle phase they - Require association regulate. with cyclins for - Cyclins are activation degraded when - CDK’s peak their function is no activity drives next longer needed. phase of cell cycle - Can be regulated by to start other regulators - increase in S phase - and has maximum activity at M phase Availability - Cyclins are - CDKs are present at synthesized and constant levels degraded at specific throughout the cell points in the cell cycle cycle. - Regulated activity, - Expression is not availability regulated and not constant ◦List the cyclin-CDK pairs involved in cell division and pair to the corresponding stage in the cell cycle - Cyclin D + CDK4, CDK6: - Increases in G1. - Cyclin E + CDK2: - Peaks at the G1/S transition. - Cyclin A + CDK1/2: - Peaks in S phase and early G2. - Cyclin B CDK1: - Peaks in G2 and M phase. Activity Availability Effect Cyclin A decreased decreased Hinder progression through S phase and the G2 phase Cyclin B decreased decreased cell will not be able to activate CDK1, transition into  mitosis will be blocked. Cyclin A increased increased promote the transition into mitosis. Cyclin A increased increased related to the S phase and the G2 phase, helping in the preparation for mitosis CDK2 decreased decreased hinder the progression through the S phase and into the G2 phase but would not be the primary factor preventing entry into mitosis 2. Assess the consequence for cellular division and/or molecular processes for changes in each of the following: binding at E2F promoter, Rb phosphorylation, cyclin availability, Cyclin/CDK activity, fatty acid metabolism, and transcription factor complexes (i.e. DREAM complex binding) ◦Identify the following molecules and match their activity to specific steps in cell cycle regulation: p53, pRb, p21, E2F and E2F promoter, cyclins, CDKs, cyclin/CDK complexes, p107/p130, fatty acid synthase, and transcription factor complexes Molecule Activity Specific steps in Cell cycle Regulation p53 - Responds to DNA - G1 phase checkpoint damage by inducing regulation cell cycle arrest or - DNA damage response. apoptosis. - Activates p21 to halt cell cycle  progression. pRb (Retinoblastoma - Binds to E2F - G1 phase, regulation of Protein) transcription factors, E2F activity, transition to inhibiting their S phase. activity. - Phosphorylation releases E2F to activate DNA replication genes. p21 - Inhibits cyclin/CDK - G1 phase, G2/M phase complexes in checkpoint response to DNA - halts progression to allow damage signals. for repair. - Activated by p53 E2F and E2F Promoter - E2F is a - Activation of S phase transcription factor genes, transition from G1 that promotes to S phase. genes necessary for DNA synthesis. - E2F promoter is regulated by pRb. Cyclins - Regulate the - Cyclin D: G1 phase. progression through - Cyclin E: G1/S transition. different phases of - Cyclin A: S and G2 phase. the cell cycle by - Cyclin B: M phase. activating CDKs. CDKs - Cyclin-dependent - G1, S, G2, and M phases; kinases that, when control cell cycle bound to cyclins, checkpoints. phosphorylate target proteins to drive cell cycle progression. Cyclin/CDK complexes - Activate CDKs to - G1/S transition phosphorylate - (Cyclin D/CDK4 substrates Cyclin E/CDK2) essential for - S phase progression through - (Cyclin A/CDK2) the cell cycle - M phase phases. - (Cyclin B/CDK1)  p107/p130 - Similar to pRb, - G1 phase, repression of these proteins can E2F activity, regulation of also bind to E2F, progression to S phase. contributing to cell cycle regulation and gene repression. Fatty Acid Synthase - Enzyme involved in - G1 phase, promotes cell fatty acid growth through increased metabolism, which lipid synthesis. can influence cell growth and division. Transcriptional Regulation via p53-mediated activation of DREAM complex - Although p53 is known to regulate gene activity through direct binding, indirect mechanisms also exist - Indirect p53-dependent repression through DREAM. - Induction of p53 leads to downregulation of genes Steps: 1. p53-mediated induction of p21 2. Induced p21 binds to and inactivates the Cyclin/CDK complex 3. Inactivation of cyclin/CDK results in a change in the phosphorylation state for p107 and p130 (pRb-related pocket proteins) 4. Hypo-phosphorylated p130/p107 facilitates DREAM complex formation 5. Transcription is repressed by DREAM displacement of activating complexes at target promoters ◦List the steps involved in E2F promoter activation and repression Steps Involved in E2F Promoter Activation and Repression: Repression: 1. pRb Binding: In G1 phase, pRb binds to E2F transcription factors, forming a complex that inhibits E2F's ability to activate genes necessary for S phase. 2. p107/p130 Binding: These proteins can also bind to E2F, further repressing its activity. Activation:  1. pRb Phosphorylation: Cyclin/CDK complexes (e.g., Cyclin D/CDK4, Cyclin E/CDK2) phosphorylate pRb, causing it to release E2F. 2. E2F Release: Once freed, E2F transcription factors bind to promoters of genes required for DNA replication and other S phase functions. 3. Transcription Activation: E2F binding to DNA promoters stimulates the transcription of genes necessary for DNA synthesis and progression to S phase ◦Match Rb phosphorylation state with corresponding activity Hypophosphorylated pRb: Activity: Binds tightly to E2F transcription factors, repressing E2F activity and preventing the transcription of genes required for DNA replication and S phase entry. State: Typically observed in early G1 phase. Hyperphosphorylated pRb: 3. Activity: Phosphorylation by Cyclin/CDK complexes causes pRb to release E2F transcription factors, allowing E2F to activate genes required for DNA replication and transition to S phase. 4. State: Occurs as the cell progresses through late G1 phase and into the S phase. 5. Evaluate the role of transcription in the regulation of cellular division ◦Explain the role of transcription factor complexes in the regulation of the cell cycle 1. Role of Transcription Factor Complexes in Cell Cycle Regulation: Transcription Factor Complexes: ○ Function: Transcription factor complexes are crucial for regulating the expression of genes that control cell cycle progression. These complexes interact with specific DNA sequences in the promoters of target genes to either activate or repress their transcription, thereby influencing various stages of the cell cycle. ○ Key Complexes: E2F Complexes: E2F transcription factors bind to promoters of genes necessary for DNA replication and S phase entry. In association with Rb (retinoblastoma protein) and its related proteins (p107, p130), E2F activity is tightly regulated. During early G1 phase, E2F is inhibited by pRb. As pRb is phosphorylated later in G1 phase, E2F is released to activate genes that drive the cell into S phase.  DREAM Complex: The DREAM complex (DP, RB-like, E2F, and MuvB) represses genes required for cell cycle progression during G1 and early S phases. It binds to specific DNA regions to prevent the transcription of genes that promote cell division until the appropriate phase of the cell cycle is reached.