23. Cell Cycle and Checkpoints

MIT OpenCourseWare

12 May 202048:19

EducationalLearning

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TLDRThe video discusses the cell division cycle and how it is regulated in cells to ensure proper replication and division. It focuses on key control systems like cyclin-dependent kinases and cyclins that mediate events during specific cell cycle phases. Checkpoints and regulated proteolysis of cyclins are also discussed as quality control mechanisms. Experiments in yeast, frogs, and cell extracts that led to discoveries about these control systems are explained, like how the destruction box on cyclins targets them for degradation. Overall, the video aims to unpack how orderly events in the cell cycle are governed to enable cells to accurately replicate and divide.

Takeaways

😀 The cell division cycle involves key events like DNA replication and cell division that must happen in a proper order
👍 Signaling pathways can promote cells to enter the cell division cycle and divide
🧬 The control system for the cell cycle involves cyclin dependent kinases (CDKs) and cyclins that regulate different cell cycle phases
⏰ Different cyclins oscillate and appear at different cell cycle phases, controlling the order of events
🔬 Genetic screens in yeast identified key cell cycle genes like CDC28, the cyclin dependent kinase
❗Checkpoints ensure quality control and proper order of cell cycle events before allowing the next event
🧪 Experiments with frog egg extracts showed cyclin proteolysis causes the oscillation
👽 A non-degradable cyclin mutant causes a mitotic arrest
🔍 The mRNA for cyclins is constant, the cyclin protein levels oscillate
💉 Growth factor signaling upregulates cyclins to promote cell cycle entry

Q & A

What are the four distinct phases of the cell division cycle?
-The four distinct phases are: S phase (DNA synthesis), M phase (mitosis), G1 phase (gap phase), and G2 phase (gap phase).
What is a cyclin dependent kinase (CDK)?
-A cyclin dependent kinase (CDK) is a kinase that can post-translationally modify other proteins by adding a phosphate group to them. This allows the CDK to modify events and control when they happen in the cell cycle.
What is the role of cyclin in the cell division cycle?
-Cyclin is the regulatory subunit of CDK. Without cyclin, CDK is inactive. The different cyclins that appear at different phases of the cell cycle activate CDK and determine its substrate specificity - i.e. which proteins CDK phosphorylates.
What is a cell cycle checkpoint?
-A checkpoint is a quality control mechanism that operates in the cell cycle to ensure that one event doesn't occur until the preceding event happens correctly. Checkpoints ensure proper order of events and ensure events are completed properly before the next event occurs.
What happens at the DNA damage checkpoint?
-At the DNA damage checkpoint, the cell cycle is delayed if DNA damage is detected or if DNA is still replicating. This delay signal inhibits cyclin CDK activity which governs the G1 to S and G2 to M transitions, slowing or halting the cell cycle.
How do cyclin levels oscillate in the cell cycle?
-Cyclin levels oscillate due to regulated proteolysis. Cyclins are tagged for degradation by the attachment of ubiquitin proteins. This targets them to the proteasome for rapid destruction, causing cyclin levels to rise and fall.
What was the purpose of using Xenopus egg extracts?
-Xenopus egg extracts provided a cell-free biochemical system that could recreate the cell cycle in vitro. This allowed researchers to definitively test the role of mRNA, mitotic cyclins, and cyclin degradation in controlling the oscillation of cyclins.
What happens in RAD9 mutants when DNA is damaged?
-In RAD9 mutants, the G2 delay after DNA damage is disrupted. The cell progresses into mitosis without delay, resulting in death of progeny cells due to the damaged DNA.
What is regulated proteolysis?
-Regulated proteolysis is the regulated degradation of proteins mediated by ubiquitination and targeting to the proteasome. It allows precise control over protein levels in the cell.
What is the role of CDC28 in budding yeast?
-CDC28 encodes the cyclin dependent kinase in budding yeast. Mutation of CDC28 causes cells to arrest in G1, unable to transition into S phase and replicate their DNA.

Outlines

00:00

😀 Overview of cell division cycle phases and key events

The cell division cycle consists of 4 phases - S phase (DNA synthesis), M phase (mitosis and chromosome segregation), G1 phase (preparation), and G2 phase (quality control). Key events that must occur include duplication of DNA and organelles so daughter cells inherit copies, cell growth, and finally cell division through mitosis. Signaling pathways and nutrient availability regulate entry into the cell cycle.

05:04

😊 Regulation of cell cycle entry and progression

A control system called cyclin dependent kinase (CDK) and cyclins ensures proper order of cell cycle events. Different cyclins bind CDK to target specific proteins for phosphorylation at different cell cycle phases. Cyclins appear in a defined order, determining which events happen when. Checkpoints like the DNA damage checkpoint inhibit CDK to delay the cycle until preceding events complete properly.

10:06

🧐 Discovery of cell cycle control machinery

Mutant screens in budding yeast identified cell division cycle (CDC) genes like CDC28, which encodes the yeast CDK. CDC28 temperature sensitive mutant causes G1 arrest, showing CDC28 is required for G1/S transition that commits cell to divide. Subsequent work showed conserved control from yeast to humans involving CDK and cyclins.

15:08

😎 Defining DNA damage checkpoint

In normal cells, DNA damage causes a G2 delay allowing repair before mitosis. Mutation of RAD9 eliminates the G2 delay after damage, resulting in death upon mitotic entry with damaged DNA. This shows RAD9 promotes the cell cycle delay that defines the DNA damage checkpoint.

20:10

🤓 Cyclin protein levels oscillate via regulated proteolysis

Cyclin levels rise and fall during particular cell cycle phases. Experiments in frog egg extracts showed constant cyclin mRNA and CDK, with protein oscillation only. Adding non-degradable cyclin mutant caused mitotic arrest from high cyclin levels, indicating cyclin proteolysis by ubiquitination causes oscillation.

Mindmap

Keywords

💡cell division cycle

The cell division cycle is the sequence of events that take place as a cell grows, replicates its DNA, segregates its chromosomes, and divides to form two daughter cells. It consists of four phases - G1, S, G2, and M phases. Understanding how the cell division cycle is regulated is crucial, as dysregulation can lead to cancer.

💡cyclin-dependent kinase (CDK)

CDK is an enzyme that phosphorylates target proteins to control events during the cell division cycle. CDK requires binding to a regulatory cyclin subunit to be activated. Different cyclin-CDK complexes appear at different cell cycle phases to phosphorylate specific targets.

💡cyclin

Cyclins are regulatory proteins that bind and activate CDK. Different cyclins oscillate in levels over the course of the cell cycle. The cyclin present determines CDK target specificity, thus governing which cell cycle events occur at different phases.

💡checkpoint

Checkpoints are control mechanisms that ensure one cell cycle event is completed properly before the next event occurs. An example is the DNA damage checkpoint, which delays the cell cycle to provide time for DNA repair before chromosome segregation.

💡regulated proteolysis

The cyclical synthesis and degradation of cyclins is mediated by regulated proteolysis. Cyclins are tagged for degradation by the addition of ubiquitin molecules. They are then targeted to and degraded by the proteasome.

💡budding yeast

Budding yeast was used in early studies of the cell division cycle. The size of the bud indicates the cell cycle phase, allowing screens for mutants arrested at specific phases. This led to the discovery of CDC28, the yeast CDK gene.

💡temperature sensitive mutant

A temperature sensitive mutant shows a phenotype only at certain temperatures. This allowed identification of cell cycle mutants in yeast - cells grew normally at 22°C but arrested at 37°C. The CDC28 mutant arrested at G1 phase.

💡G1/S transition

The G1 to S phase transition is the commitment point where cells enter the division cycle. G1 cyclin-CDK activity mediates this transition. The CDC28 gene in yeast encodes the CDK needed to pass this restriction point.

💡RAD9

The RAD9 gene mediates the G2 delay after DNA damage, allowing time for repair. Mutation of RAD9 caused cells to progress into mitosis with damaged DNA, resulting in death of progeny cells.

💡destruction box

The destruction box is a protein sequence targeted for ubiquitination and degradation. Cyclins with mutant destruction boxes failed to degrade in frog egg extracts, causing mitotic arrest.

Highlights

The cell division cycle consists of four distinct phases - S phase, M phase, G1 phase, and G2 phase

Cyclin-dependent kinases (CDKs) and cyclins are key components regulating the cell division cycle

Different cyclins bind to CDKs at specific cell cycle phases, conferring substrate specificity

CDC28 was identified in yeast as the gene encoding cyclin-dependent kinase, conserved across species

Checkpoints ensure proper order of cell cycle events by delaying progression until preceding events complete correctly

The RAD9 gene in yeast is involved in the DNA damage checkpoint, promoting a cell cycle delay for repair

Ubiquitination targets proteins like cyclins for degradation, causing their oscillating levels during cell cycle

Xenopus egg extracts can replicate the cell cycle in vitro, allowing analysis of specific components

M-cyclin mRNA levels remain constant, protein levels oscillate due to regulated proteolysis

Non-degradable cyclin mutants cause mitotic arrest, indicating importance of cyclin destruction

G1 phase - cell decides whether to enter the cell cycle based on growth signals

S phase - DNA is replicated by proteins like helicases and polymerases

M phase - sister chromatids separate to daughter cells via the mitotic spindle

G2 phase - quality control checkpoint ensures DNA replication before mitosis

Cyclins activate CDKs to phosphorylate specific substrates at different cell cycle phases

Transcripts

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