UNIT 10 – Cell Cycle Regulation (Q&A) | MZO-001 MSCZOO | IGNOU

SAQ 1

a) The mitotic apparatus, also known as the ................... apparatus, plays an important role in chromosome segregation during cell division.

Answer: spindle

b) During interphase, the cell undergoes growth and performs its regular functions in the .................... phase.

Answer: G1 (Gap 1)

c) The cell cycle is highly regulated by a group of proteins called ................... .

Answer: cyclins

d) The ...................... checkpoint in the cell cycle ensures that DNA is undamaged before progressing to mitosis.

Answer: G1/S

e) What are the key components of the mitotic apparatus?

The mitotic apparatus is a temporary but essential structure formed during mitosis, especially during the metaphase and anaphase stages. It plays a critical role in the proper alignment and separation of chromosomes into daughter cells. The key components of the mitotic apparatus are mainly three and each part performs a specific function to ensure accurate chromosome segregation.

There are three key components of the mitotic apparatus:

1. Spindle Fibres (Microtubules):

These are the main structural elements of the mitotic apparatus. Spindle fibres are made up of microtubules, which are dynamic protein filaments composed of tubulin. They originate from the centrosomes or spindle poles and extend toward the center of the cell. These fibres attach to chromosomes at the centromere region through the kinetochore and help pull the chromatids apart during anaphase. There are three types of spindle fibres: kinetochore microtubules, polar microtubules and astral microtubules.

1. Kinetochore Microtubules: These attach to the kinetochore region of chromosomes and are directly involved in pulling chromatids apart.
2. Polar Microtubules: These interact with microtubules from the opposite pole and help in pushing the poles apart.
3. Astral Microtubules: These radiate outward from the centrosomes to help position the spindle inside the cell.

[Note: Kinetochore microtubules and kinetochores are not the same. Kinetochore microtubules are a subtype of spindle fibres. They are thread-like fibres that come from the spindle pole and connect to the kinetochore. On the other hand, the kinetochore is the protein patch found on the centromere of a chromosome.]

2. Centrosomes (Spindle Poles):

Centrosomes are the organizing centres for spindle formation. Each centrosome contains a pair of centrioles surrounded by pericentriolar material. They duplicate before mitosis and move to opposite poles of the cell during prophase. Centrosomes help in the nucleation and organization of microtubules, which form the spindle fibres.

3. Kinetochores:

These are disc-shaped protein structures located at the centromere of each chromosome. The kinetochore serves as the attachment site for spindle microtubules. It is also involved in sensing tension and helping to regulate the movement of chromosomes during metaphase and anaphase. It plays an active role in ensuring that each sister chromatid is attached to the correct spindle pole.

SAQ 2

a) Give another name of the G1 phase?

Another name for the G1 phase is the "Gap 1 phase" or "First gap phase."

b) Discuss cellular activities occur during the G1 phase?

The G1 phase (Gap 1 phase) is the first and one of the most critical stages of interphase, which occurs after M phage (mitosis) and before DNA replication in the S phase. This phase plays a pivotal role in preparing the cell for the upcoming processes of DNA synthesis and cell division. During G1, the cell undergoes essential growth and various metabolic activities to ensure it has the necessary resources for DNA replication. The G1 phase also serves as a checkpoint to assess the cell's readiness to move to the next phase of the cycle. It is a time when the cell grows in size, synthesizes proteins and checks for DNA integrity, laying the foundation for successful cell division.

Here is the important cellular activities that occur during the G1 phase:

1. Cell Growth:

During the G1 phase, the cell undergoes substantial growth. The cell increases in size as it synthesizes proteins and organelles needed for its function. This growth ensures that the daughter cells have enough resources to perform their normal functions and to prepare for DNA replication in the S phase.

2. Protein and RNA Synthesis:

The cell produces a variety of proteins, including those required for DNA replication and cell division, as well as enzymes necessary for cellular metabolism. RNA synthesis also occurs in the G1 phase, contributing to the production of various enzymes and structural proteins needed for the next stages of the cell cycle.

3. Organelle Duplication:

In G1, the cell begins to duplicate its organelles. For example, the number of mitochondria increases to provide energy for the cell's upcoming functions. Ribosomes and other organelles like the endoplasmic reticulum (ER) also undergo increased production to support the growing cell.

4. Activation of Cyclins and Cyclin-Dependent Kinases (CDKs):

The progression through the G1 phase is highly regulated by specific proteins called cyclins and cyclin-dependent kinases (CDKs). These proteins help the cell transition from G1 to the S phase, ensuring that DNA replication occurs only when the cell is ready. The G1 checkpoint is crucial in deciding whether the cell proceeds to the S phase or enters a G₀ phase (resting phase).

5. Increase Metabolic Activity:

During G1, the cell actively engages in metabolic processes such as glycolysis, energy production and biosynthesis. This increased metabolism supports the growth of the cell and prepares it for the upcoming replication of its DNA.

6. Preparation for DNA Replication:

Although DNA replication does not occur during G1, it is during this phase that the cell prepares for the S phase. The cell synthesizes essential enzymes like DNA polymerases and other factors involved in DNA replication. The G1 phase is also critical for DNA repair, ensuring that any damage to the genome is repaired before replication begins.

7. Checking for DNA Damage:

The cell monitors its DNA for any damage during G1. If any damage is detected, the cell can halt progression to the S phase through checkpoint control mechanisms. This helps in preventing the replication of damaged DNA, thereby protecting the genome's integrity. If the damage is irreparable, the cell may undergo apoptosis (programmed cell death).

8. Decision to Enter S Phase or G₀ Phase (Resting Phase):

At the end of the G1 phase, the cell faces a crucial decision at the restriction point. The cell will either enter the S phase for DNA replication, or it may enter a quiescent state called as G₀ phase (resting phase). In G₀ phase, the cell is temporarily or permanently inactive, not preparing for division, depending on the organism and cell type. This decision is influenced by external signals like growth factors, nutrients and the overall health of the cell.

c) What is the purpose of G1 checkpoint?

The G1 checkpoint, also known as the restriction point, is a critical control mechanism in the G1 phase of the cell cycle. Its main purpose is to make sure that the cell is fully prepared before it enters the S phase, where DNA replication takes place.

Purpose of the G1 Checkpoint:

The main goal of the G1 checkpoint is to check whether the cell is in a proper condition to continue dividing. If the conditions are not right, the cell will not proceed to the next phase. Instead, it may pause for repair or even enter a resting phase called G₀ phase.

The key functions of the G1 checkpoint are:

1. Check for DNA damage:

• The checkpoint ensures that the cell's DNA is not damaged. If the DNA has any errors or breaks, the cell activates repair mechanisms. If the damage is beyond repair, the cell may go through apoptosis (programmed cell death) to prevent passing the error to daughter cells.

2. Check for cell size and growth:

• The checkpoint verifies that the cell has grown to a sufficient size and has enough cytoplasm and organelles to support two daughter cells after division.

3. Check for sufficient energy and nutrients:

• The cell must have enough ATP and nutrients (like amino acids and glucose) to carry out the energy-demanding processes of DNA replication and cell division.

4. Check for proper external signals:

• Some cells only divide when they receive proper signals from outside, like growth factors. The G1 checkpoint confirms whether these signals are present.

d) Which proteins regulate the progression from G1 to S phase?

In eukaryotic cells, the transition from G1 to S phase of the cell cycle is one of the most important and carefully regulated steps. This phase decides whether a cell will proceed toward DNA replication or stay in a resting state (G₀ Phase) or even go for repair. Cells make this decision based on internal and external signals, and this entire decision making process is controlled by specific regulatory proteins.

There are five main types of protein regulators that play a central role in G1 to S phase transition. These proteins work in coordination and any disturbance in their function can lead to abnormal cell division or diseases like cancer.

1. Cyclins (Cyclin D and Cyclin E)

Cyclins are regulatory proteins that appear and disappear during different phases of the cell cycle. During the early G1 phase, Cyclin D is produced in response to external signals like growth hormones or mitogens. It binds to CDK4 and CDK6, forming active complexes that start phosphorylating various target proteins and push the cell forward in the G1 phase.

Later, in the late G1 phase, Cyclin E starts getting expressed. It binds to CDK2 and this Cyclin E-CDK2 complex is very important for the actual commitment of the cell to enter S phase. Without the Cyclin E-CDK2 complex, the cell cannot cross the G1 checkpoint.

2. Cyclin-Dependent Kinases (CDKs)

CDKs are enzymes that need to bind with cyclins to become active. They do not function alone. The main CDKs active in the G1 to S transition are CDK4, CDK6 and CDK2.

• CDK4 and CDK6 become active after binding with Cyclin D

• CDK2 becomes active after binding with Cyclin E

These active CDK-cyclin complexes phosphorylate target proteins, mainly the retinoblastoma protein (Rb), which acts like a brake on the cell cycle.

3. Retinoblastoma Protein (Rb)

The retinoblastoma protein (Rb) protein is a tumour suppressor protein. In its original, unphosphorylated form, Rb binds to a transcription factor called E2F and prevents it from activating genes required for DNA replication.

Once Rb is phosphorylated by the CDK4/6-Cyclin D and CDK2-Cyclin E complexes, it undergoes a change in shape and releases E2F. This release is the key signal that allows the cell to move from G1 into S phase and begin DNA synthesis.

4. E2F Transcription Factor

E2F plays a very important role in initiating the S phase. Once freed from Rb, it starts the transcription of many genes that are essential for the S phase. These include genes for DNA polymerases, nucleotide synthesis enzymes and histone proteins.

Without active E2F, the S phase cannot begin. Thus, E2F acts like a master switch that turns on the machinery needed for DNA replication.

5. CDK Inhibitors (CKIs)

These proteins work as brakes or checkpoints. If the cell has DNA damage or is not ready for replication, CDK inhibitors will stop the activity of CDK-cyclin complexes. Some common CKIs are p21, p27 and p16.

For example, in response to DNA damage, the protein p53 activates the transcription of p21, which then binds to and inhibits the Cyclin E-CDK2 complex, stopping the cell from entering S phase. This gives the cell time to repair itself before continuing.

e) What happens at the S phase of the cell cycle?

The S phase, also known as the Synthesis phase, is the second stage of interphase in the eukaryotic cell cycle. It comes after the G1 phase and before the G2 phase. The most important event of the S phase is DNA replication, which means the cell copies its entire genetic material so that each daughter cell can receive an exact copy of the genome after cell division.

In G1 phase, the cell has a normal diploid set of chromosomes. But during the S phase, each chromosome replicates to form two identical sister chromatids, which are attached at a central region called the centromere. However, the number of chromosomes does not change. For example, a human cell has 46 chromosomes before and after the S phase, but the DNA content doubles from 2C to 4C. This increase is crucial because without complete replication, cells cannot divide properly.

DNA replication begins at specific points on the DNA called origins of replication. Multiple origins are used simultaneously to speed up the process in eukaryotic cells. Several important enzymes and proteins are involved:

• DNA helicase: unwinds the DNA helix

• Single-stranded binding proteins (SSBPs): keep the DNA strands open

• Primase: adds RNA primers

• DNA polymerase: adds new complementary DNA bases

• DNA ligase: joins Okazaki fragments on the lagging strand

Besides DNA, the centrosome also duplicates during this phase. The centrosome contains the centrioles and is essential for forming the spindle apparatus in mitosis or meiosis.

The S phase is highly regulated and multiple checkpoints monitor whether DNA replication is proceeding correctly. If any error or damage is detected, the cell can stop the cycle to repair the problem before continuing to the G2 phase.

In this way, the S phase ensures that each new cell receives a full and accurate set of genetic information. Mistakes in this phase can lead to mutations or chromosomal abnormalities, which may result in diseases like cancer.

f) What is the significance of the G2 phase in the cell cycle?

The G2 phase (also known as Gap 2 phase) is the third and final phase of interphase in the cell cycle. It occurs after DNA replication in the S phase and before the cell enters mitosis (M phase). While the G2 phase may seem like a resting phase, it is actually a highly active period during which the cell prepares for division. The G2 phase ensures that the cell has successfully completed DNA replication and is ready to divide. It also serves as a checkpoint for the cell to correct any errors that may have occurred during DNA replication, ensuring that only healthy, complete genetic material is passed on to daughter cells.

1. DNA Damage Check and Repair through the G2-M Checkpoint

One of the major significances of the G2 phase is to check for DNA damage that may have occurred during the S phase, where DNA replication takes place. If there is any damage in the DNA, the cell does not immediately move into mitosis. Instead, a special control system known as the G2-M checkpoint becomes active. This checkpoint acts like a security gate. It carefully checks the DNA for errors or damage. If any damage is found, it stops the cycle temporarily and allows time for repair enzymes to fix the problem. This checking is mainly regulated by proteins like cyclins and CDKs. Only when the DNA is fully repaired and safe, the checkpoint gives permission to enter mitosis. But if the damage is too serious and cannot be fixed, then the cell activates a self-destruct process called apoptosis, which prevents the damaged cell from dividing and protects the body from harmful mutations.

2. Final Growth and Protein Synthesis

During the G2 phase, the cell continues to grow in size and produces all the proteins and enzymes that are needed for mitosis. These include tubulin for spindle fibres, proteins for chromosome condensation, and energy molecules like ATP. This helps the cell get physically ready for the division process.

3. Centrosome Duplication and Spindle Formation

In the G2 phase, centrosomes (the organizing centers for microtubules) replicate. This is crucial for the formation of the mitotic spindle during mitosis. The spindle fibers will be responsible for ensuring that chromosomes are properly separated and distributed to the daughter cells. Without proper centrosome duplication, the spindle cannot form correctly, leading to errors in chromosome segregation.

4. Ensures Genetic Stability

By checking and repairing DNA and only allowing healthy cells to enter mitosis, the G2 phase ensures that each daughter cell will have the correct genetic material. This protects the organism from mutations, cancer and other problems related to faulty cell division.

g) What is the purpose of the G2 checkpoint?

The G2 checkpoint is often referred to as the G2-M checkpoint because it is located at the boundary between the G2 phase and the M phase (mitosis) of the cell cycle. This checkpoint plays a key role in deciding whether the cell is ready to move from G2 phase into mitosis. The name "G2-M" itself reflects its position and function, it lies at the end of the G2 phase but directly regulates the cell's entry into the M phase.

During the G2 phase, the cell completes the synthesis of proteins and prepares all necessary components for mitosis. However, before entering mitosis, the cell must ensure that the DNA, which was replicated during the S phase, is completely and correctly duplicated and that there is no DNA damage. At this critical point, the G2-M checkpoint becomes active. It functions like a gatekeeper or quality control system. It checks:

• Whether DNA replication has been completed without any errors
• Whether the DNA is free of any damage
• Whether the cell has all resources and proteins necessary to undergo mitosis

This checkpoint prevents the cell from prematurely entering mitosis. If any issues are found, such as incomplete replication or DNA damage, then the checkpoint halts the progression of the cell cycle. This delay provides time for repair mechanisms to fix the issues. If the damage is not repairable, the cell may undergo apoptosis, which is a programmed cell death process, to prevent the division of cells with defective genetic material.

This whole process is regulated by several important proteins. ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related) are two proteins that detect DNA damage. When damage is detected, they activate Chk1 and Chk2 kinases. These kinases then stop the function of Cdc25 phosphatase, which is needed to activate Cyclin B-CDK1 complex. Without this complex, the cell cannot enter mitosis. This is how the checkpoint ensures that the cell cycle only continues when it is safe to do so.

Thus, the checkpoint is crucial for maintaining genomic integrity, and it is referred to as the G2-M checkpoint because this checkpoint lies between G2 and M phases and controls the transition between them, scientists call it the G2-M checkpoint rather than just the G2 checkpoint.

h) What happens when DNA damage is detected during the G2 phase?

When DNA damage is detected during the G2 phase, the cell activates a control mechanism known as the G2-M checkpoint. This checkpoint prevents the cell from entering mitosis until the damage is repaired. The proteins ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related) first detect the DNA damage. They then activate Chk1 and Chk2 kinases, which block the activity of Cdc25 phosphatase. Normally, Cdc25 activates the Cyclin B-CDK1 complex, which is essential for starting mitosis. But when Cdc25 is inhibited, the Cyclin B-CDK1 complex stays inactive, so the cell cannot move forward. This delay gives time for DNA repair enzymes to fix the damage. If the repair is successful, the cell enters mitosis. However, if the damage is too severe and cannot be fixed, the cell activates apoptosis (programmed cell death) to destroy itself and avoid passing damaged DNA to new cells.

SAQ 3

a) What are cyclins?

Cyclins are a special group of regulatory proteins that control the proper timing and order of the cell cycle. They are called "cyclins" because their concentration inside the cell rises and falls in a cyclical pattern during different phases of the cell cycle. They are not enzymes by themselves but become functional when they bind with another group of proteins called cyclin-dependent kinases (CDKs). These CDKs are enzymes that phosphorylate target proteins and this phosphorylation triggers specific events in the cell cycle like DNA replication or mitosis.

Cyclins are essential because they ensure that the cell cycle progresses in the correct sequence. Without them, cells would not know when to grow, when to copy DNA, or when to divide. Cyclins are produced at specific times during the cell cycle and once their function is complete, they are broken down by a system called the ubiquitin-proteasome pathway. This degradation prevents the cell from repeating the same phase and forces it to move forward in the cycle.

The balance of cyclins and CDKs is highly regulated. If cyclins are not produced or destroyed properly, it may lead to cell cycle problems, uncontrolled cell division, or even cancer. So, cyclins not only help the cell grow and divide at the right time but also protect it from making dangerous mistakes.

Types of Cyclins

There are four major types of cyclins, and each one works at a particular phase of the cycle:

1. Cyclin D (Active in G1 phase):

Cyclin D is the first cyclin to appear during the cell cycle. It is produced in response to external growth signals. Its main job is to help the cell pass the G1 checkpoint and move forward. It binds with CDK4 and CDK6, and this complex helps in the phosphorylation of the retinoblastoma (Rb) protein, which in turn releases E2F transcription factors, promoting entry into the S phase. Cyclin D acts like a sensor of growth signals and prepares the cell to commit to division.

2. Cyclin E (G1 to S transition):

Cyclin E is expressed later in the G1 phase and is very important for the G1/S transition. It binds with CDK2 and this complex helps in the initiation of DNA replication. Cyclin E ensures that all preparations for DNA synthesis are complete and that the cell is now ready to enter the S phase. It also helps the cell to pass through the restriction point, after which the cell becomes committed to division.

3. Cyclin A (S and G2 phase):

Cyclin A is active during both the S and G2 phases. It first binds with CDK2 in the S phase to promote DNA replication, ensuring that the entire genome is copied once and only once. Later, in the G2 phase, it switches to bind with CDK1 and helps prepare the cell for mitosis. Cyclin A helps regulate both DNA synthesis and the start of mitotic events.

4. Cyclin B (G2 to M transition):

Cyclin B is the last major cyclin in the cycle. It binds with CDK1 to form the well-known Maturation Promoting Factor (MPF), which is essential for the entry into mitosis. MPF triggers major events of mitosis like chromatin condensation, nuclear envelope breakdown, and spindle formation. Cyclin B accumulates in the G2 phase and reaches its peak during mitosis.

b) How are cyclins named?

Cyclins are named based on their cyclical pattern of appearance and disappearance during the cell cycle and the specific phases in which they are active. The term "cyclin" was first introduced by Tim Hunt, a British scientist, who discovered a protein in 1982 in sea urchin embryos that increased and decreased in regular cycles during cell division. Because of this repeating pattern, he casually named the protein "cyclin" to reflect its cyclic behaviour. His research was officially published in 1983 and later he was awarded the Nobel Prize in Physiology or Medicine in 2001, along with Paul Nurse and Leland Hartwell, for their work on cell cycle regulation.

After this discovery, more cyclins were identified in other organisms. As a result, they were systematically named using letters like Cyclin A, B, C, D, and E. This naming usually depends on either the order in which they were discovered or the cell cycle phase in which they are most active.

For example:

• Cyclin D: Appears in early G1 phase

• Cyclin E: Functions during G1 to S phase transition

• Cyclin A: Active in S and G2 phases

• Cyclin B: Works at G2/M transition and helps in starting mitosis

This alphabetical system helps in identifying which cyclin is working at which stage of the cell cycle. Some cyclins are also named based on their structural features, function, or association with specific CDKs (cyclin-dependent kinases).

Thus, the naming of cyclins reflects a combination of their discovery timeline, functional role and cyclical behaviour in the cell division process.

c) What is the role of cyclins in the cell cycle?

Cyclins are regulatory proteins that play a very important role in the regulation of the cell cycle. They are a special group of regulatory proteins that control the proper timing and order of different phases of the cell cycle, such as G1, S, G2 and M phase. Cyclins do not work alone. They bind with specific enzymes called Cyclin-Dependent Kinases (CDKs) to form an active cyclin-CDK complex. This complex helps in starting and controlling various steps of the cell cycle.

The levels of cyclins are not constant. They rise and fall at specific times during the cycle. This timely appearance and disappearance of cyclins ensure that the cell cycle proceeds in a proper sequence.

Cyclins Have the Following Roles in the Cell Cycle:

1. Regulation of Phase Transitions:

Cyclins regulate the transition of the cell from one phase to another. Each cyclin activates a specific CDK complex that is required for the progression to the next phase. For example, Cyclin D binds with CDK4/6 during the G1 phase to help the cell transition into the S phase. Similarly, Cyclin E-CDK2 activates the necessary proteins for DNA replication in the S phase and Cyclin B-CDK1 is essential for entering mitosis during the G2-M transition.

2. Checkpoint Control and DNA Damage Response:

Cyclins also help maintain the integrity of the cell's DNA. Before the cell progresses to the next phase, certain cyclins ensure that the DNA is intact and free from damage. Cyclins like Cyclin A and Cyclin B regulate checkpoints at various stages (such as G1, G2 and during mitosis). If DNA damage is detected, the cell cycle is temporarily paused to allow for repair. If the damage is irreparable, cyclins help activate apoptosis (programmed cell death) to prevent the damaged DNA from being passed on.

3. Activation of CDK Complexes:

The primary function of cyclins is to activate Cyclin-Dependent Kinases (CDKs). When a cyclin binds to a CDK, it forms an active complex capable of phosphorylating specific target proteins. This phosphorylation drives the cell cycle forward by triggering various processes needed for the transition between different phases. For example, Cyclin D-CDK4/6 complexes help the cell pass through the G1 checkpoint, while Cyclin A-CDK2 complexes facilitate the progression through S phase.

4. Degradation After Function Completion:

After a cyclin has performed its regulatory function, it must be degraded to maintain the timing of the cell cycle. This is achieved through ubiquitination, a process where the cyclin is tagged for degradation by a proteasome. This degradation ensures that cyclins are only active when needed and prevents the cell cycle from moving forward unchecked.

d) Give examples of cyclins and their association with cell cycle phases.

Examples of Cyclins and Their Association with Cell Cycle Phases:

1. Cyclin D: This is associated with the G1 phase.
2. Cyclin E: This is associated with the G1 to S phase transition.
3. Cyclin A: This is associated with the S and G2 phases.
4. Cyclin B: This is associated with the M phase. 

e) Why is the dynamic regulation of cyclins important?

Cyclins are essential regulatory proteins that control the progression of the cell cycle. Their activity is highly regulated in a dynamic manner to ensure the cell cycle proceeds at the appropriate time and under the right conditions. This regulation involves the synthesis, activation and degradation of cyclins at specific points during the cycle. The precise control of cyclin levels and activity is crucial for maintaining normal cell function and preventing diseases like cancer. The dynamic regulation of cyclins helps coordinate various processes such as DNA replication, cell division and checkpoint control, ensuring the cell divides only when it is ready and the conditions are appropriate.

Importance of Dynamic Regulation of Cyclins:

1. Controlled Progression through the Cell Cycle:

Cyclins regulate the transition of cells from one phase of the cell cycle to the next by activating Cyclin-Dependent Kinases (CDKs). The dynamic regulation of cyclins ensures that the cell proceeds from one phase to another at the appropriate time. For example, Cyclin D is produced at the start of the G1 phase and helps the cell transition into the S phase, while Cyclin B is essential for the cell to enter mitosis. If cyclin levels are not highly regulated, the cell cycle may advance too quickly or too slowly, leading to developmental defects or abnormal growth.

2. DNA Replication and Repair:

Cyclins also play a role in ensuring that the cell only proceeds to the next phase when DNA replication has been completed and when any DNA damage has been repaired. The dynamic regulation of cyclins ensures that the checkpoints in the cell cycle are functional. For example, Cyclin E-CDK2 helps control the S phase to ensure that DNA replication occurs correctly, while Cyclin A-CDK2 monitors DNA damage repair. If cyclin levels are not properly controlled, the cell could replicate damaged DNA, leading to mutations or genomic instability.

3. Prevention of Uncontrolled Cell Division:

One of the most crucial aspects of cyclin regulation is preventing uncontrolled cell division, which can lead to cancer. Cyclins must be degraded at the correct time after they have completed their function. For instance, after Cyclin B activates the Cyclin B-CDK1 complex to enter mitosis, it is rapidly degraded to allow the cell to complete mitosis and prepare for the next cycle. If cyclins are not degraded at the proper time, the cell may stay in mitosis longer than needed, which can result in aneuploidy (abnormal chromosome number) and uncontrolled proliferation.

4. Timing and Coordination with External Signals:

The dynamic regulation of cyclins is also important in coordinating the cell cycle with external signals. For example, growth factors may stimulate the synthesis of Cyclin D, which triggers the transition from G1 to S phase. Without this regulation, cells might enter the cycle without proper external cues, which can lead to abnormal growth and development. Cyclin levels ensure that the cell responds appropriately to internal and external stimuli, ensuring proper cell division and function.

f) What are CDKs?

Cyclin-dependent kinases (CDKs) are crucial regulatory enzymes that control the progression of the cell cycle. These kinases ensure that the cell moves through the various phases of the cell cycle at the right time and in a regulated manner. However, CDKs are inactive on their own. They must bind to specific proteins called cyclins to become active. Once activated, the CDK-cyclin complex can phosphorylate target proteins, which are necessary for key processes like DNA replication, mitosis and cell division.

Role of CDKs in the Cell Cycle:

1. Regulation of Cell Cycle Phases:

CDKs play a significant role in helping the cell transition smoothly between different stages of the cell cycle. For example, CDKs facilitate the transition from G1 to S phase, where DNA replication begins, and from G2 to M phase, where the cell prepares for mitosis.

2. DNA Replication:

During the G1 phase, CDKs activate essential proteins involved in DNA replication. This ensures that the DNA is accurately copied before the cell divides. If DNA replication is incomplete or faulty, the CDK-cyclin complexes can pause the cell cycle to allow repair processes to occur.

3. Mitosis and Cell Division:

CDKs are also involved in regulating the M phase of the cell cycle. They help activate proteins that control critical mitotic processes, such as chromosome condensation, spindle formation, and the separation of chromatids, ensuring proper cell division.

4. Cell Cycle Checkpoints:

CDKs are involved in checkpoint control, ensuring that the cell cycle only proceeds when it is safe. For instance, they prevent the cell from entering the M phase if DNA replication is not complete or if there is any DNA damage. If problems are detected, CDKs can activate repair mechanisms or induce cell death to prevent the division of damaged cells.

g) How do CDKs control the cell cycle?

Cyclin-dependent kinases (CDKs) are regulatory enzymes that work as master controllers of the cell cycle. They do not act alone but get activated only when they bind with cyclins. Once active, CDKs control the flow of the cell cycle by turning "on" or "off" many important steps through phosphorylation. Their main role is to make sure that each phase of the cell cycle starts only when the cell is ready.

Now let us understand how exactly CDKs control the cell cycle. There are four main ways by which CDKs control it:

1. Phase-Specific Activation

Each CDK becomes active only in a particular phase by binding with a specific cyclin. For example, CDK4/6 binds with cyclin D in G1 phase to push the cell forward. Similarly, CDK2 binds with cyclin E for G1 to S transition and CDK1 with cyclin B helps in entry into mitosis. This ensures that CDKs only activate the next phase when the current phase is complete.

2. Phosphorylation of Target Proteins

Once CDKs are activated, they add phosphate groups to specific target proteins. These phosphorylated proteins then start different activities like DNA replication in S phase, chromosome condensation in M phase, or breakdown of nuclear membrane. Without CDK activity, these events cannot begin.

3. Regulation Through Checkpoints

CDKs help maintain checkpoint control. If DNA damage or incomplete replication is detected, CDKs are inhibited by checkpoint proteins. This stops the cell from moving to the next phase. In this way, CDKs help prevent division of damaged cells and protect from mutation.

4. CDK Inhibition and Cyclin Degradation

CDK activity is also controlled by inhibitors like p21, p27 and by removal of cyclins. When cyclins are broken down after completing their function, CDKs become inactive. This prevents unwanted activation and keeps the cycle strictly regulated.

h) Name some CDKs involved in cell cycle regulation in animals.

Cyclin-dependent kinases (CDKs) are crucial regulatory enzymes that play a key role in regulating the cell cycle in animal cells. They are called "dependent" because their activity is not independent. These kinases need to be activated by binding with another group of proteins known as cyclins. This interaction allows CDKs to regulate the progression of the cell cycle, ensuring that each phase happens at the right time and in the right sequence. The cell cycle is highly controlled to prevent errors in cell division, which could lead to diseases such as cancer.

Main CDKs Involved in Cell Cycle Regulation

There are four main types of CDKs involved in cell cycle regulation in animals: CDK1, CDK2, CDK4 and CDK6. Each of these CDKs has its own specific role and they function at different stages of the cell cycle. Their activation is crucial for the cell to progress through the various phases of growth, DNA replication and division.

Role of CDK1

CDK1 is one of the most important CDKs and is critical for the progression of the cell cycle. It works during the G2 to M phase transition. In this phase, CDK1 binds with Cyclin B, forming the Cyclin B-CDK1 complex. This complex is the primary driver of mitosis (M phase). When activated, CDK1 helps in chromosome condensation, spindle formation and the breakdown of the nuclear envelope, all necessary steps for cell division. The proper activation of CDK1 ensures that mitosis proceeds smoothly and without it, the cell cannot enter or progress through mitosis.

Role of CDK2

CDK2 is mainly involved in the G1 to S phase transition and also plays a role in the S phase itself. In the late G1 phase, CDK2 binds with Cyclin E to form a complex that allows the cell to move into the S phase, where DNA replication occurs. Once in the S phase, CDK2 continues to function by binding with Cyclin A, facilitating the progression of DNA replication. This makes CDK2 essential for the replication of genetic material, ensuring that the cell is ready to divide.

Role of CDK4 and CDK6

CDK4 and CDK6 are active mainly in the early G1 phase. These CDKs form complexes with Cyclin D and together they begin the process of cell cycle progression. Their main task is to phosphorylate the retinoblastoma protein (Rb), which normally acts as a brake on the cell cycle. Phosphorylation of Rb leads to the release of the E2F transcription factor, which then activates the genes required for DNA synthesis. This allows the cell to proceed from the G1 phase into the S phase. The activity of CDK4 and CDK6 is especially important in regulating the cell's response to external signals, such as growth factors.

i) What is the role of CDK7?

Cyclin-dependent kinase 7 (CDK7) is a vital regulatory protein in cells that plays a significant role in regulating both the cell cycle and gene expression. It is a member of the cyclin-dependent kinase (CDK) family, which means it works together with other proteins called cyclins to control the timing of cell cycle events. CDK7 is also part of a complex known as CDK-activating kinase (CAK), which activates other CDKs, allowing the cell to progress through its various stages. Besides its important role in the cell cycle, CDK7 also helps control transcription, the process by which cells make RNA copies of genes. This makes CDK7 an essential player in both cell division and gene regulation, ensuring that the cell functions properly and responds to its environment.

Role of CDK7 in Cell Cycle Regulation

CDK7 plays an important role in the regulation of the cell cycle, helping cells transition from one phase to another. Its main function is to activate other CDKs, which are needed to move the cell forward through the different stages of the cycle.

1. Activation of Other CDKs

• One of the key functions of CDK7 is to activate other cyclin-dependent kinases like CDK1, CDK2, CDK4 and CDK6. It achieves this by phosphorylating them, which means adding a phosphate group to them, a step that is necessary for their activation. These activated CDKs are responsible for controlling the transition between different phases of the cell cycle, such as from G1 to S phase (DNA replication) and from G2 to M phase (mitosis). Without CDK7's activation of these other CDKs, the cell would not be able to progress properly through the cycle, which could result in cell cycle arrest or genomic instability.

2. Ensuring Controlled Cell Cycle Progression

• By activating the necessary CDKs, CDK7 ensures that the cell progresses through the cell cycle at the right time and in an orderly manner. This control is crucial because improper progression can lead to genetic mutations, cancer, or other cell dysfunctions. CDK7's activity ensures that the cell only moves on to the next stage of the cycle when it is ready, protecting the cell from potential damage.

Role of CDK7 in Transcription Regulation

Apart from its role in the cell cycle, CDK7 is also involved in transcription regulation. It is part of a larger complex called TFIIH (Transcription factor II H), which is necessary for RNA polymerase II to transcribe genes into messenger RNA (mRNA).

1. Phosphorylation of RNA Polymerase II

• CDK7, as part of TFIIH, phosphorylates the C-terminal domain (CTD) of RNA polymerase II. This modification is critical for the transition from transcription initiation to elongation, allowing RNA polymerase II to continue the process of gene transcription. Without this phosphorylation, transcription would be stuck at the initial stage, preventing the cell from producing necessary proteins.

2. Regulation of Gene Expression

• By regulating RNA polymerase II, CDK7 controls the expression of genes. This is important because gene expression is responsible for making proteins that are crucial for cell functions, such as growth, response to stress and repair. By ensuring proper transcription, CDK7 helps the cell respond to changes in its environment and maintain normal function.

j) Give examples of cyclin B1 localisation.

Cyclin B1 plays a crucial role in the regulation of the cell cycle, specifically during the transition from the G2 phase to the M phase (mitosis). Its proper localization within the cell is essential for accurate cell division. Cyclin B1 is mainly localized to the cytoplasm during interphase and its levels gradually increase as the cell approaches mitosis. At the onset of mitosis, cyclin B1 translocates to the nucleus, where it associates with Cdk1 (Cyclin-dependent kinase 1), forming the Cyclin B1-Cdk1 complex that drives the cell into mitosis.

Here are some examples of Cyclin B1 localization:

1. Cytoplasmic Localization in Interphase

• During the interphase (G1, S, and G2 phases) of the cell cycle, cyclin B1 is primarily localized in the cytoplasm. It is sequestered in the cytoplasm and is kept inactive, preventing premature entry into mitosis. Cyclin B1 is synthesized throughout the cell cycle but remains cytoplasmic until the G2 phase.

2. Nuclear Translocation at Mitosis Onset

• As the cell enters prophase, cyclin B1 translocates from the cytoplasm to the nucleus, where it forms a complex with Cdk1. This localization is critical for the activation of Cdk1, which initiates key mitotic events, such as nuclear envelope breakdown and chromosome condensation.

3. Nuclear Envelope

• During prophase and prometaphase, cyclin B1 is localized in the nucleus, where it associates with Cdk1 to form the cyclin B1-Cdk1 complex. This complex is responsible for driving the cell into mitosis by activating proteins that facilitate chromatin condensation and spindle formation.

4. Cytoplasmic Retention during G2 and Early Mitosis

• In some cases, cyclin B1 is retained in the cytoplasm even after the cell begins mitosis. This retention is highly regulated by proteins like 14-3-3, which bind to cyclin B1 and prevent its nuclear import until the right moment. This helps ensure that mitosis is initiated only when conditions are correct.

5. Nuclear Export

• After mitosis is complete, cyclin B1 is degraded, and its levels drop. The nuclear export of cyclin B1 is also regulated, ensuring that it exits the nucleus after mitosis and is degraded, thus inactivating the Cdk1 complex and allowing the cell to exit mitosis and enter the G1 phase.

6. Spindle Localization

• During metaphase, cyclin B1 is often localized to the spindle apparatus. It plays a role in the coordination of spindle function and chromosome alignment, which is crucial for the accurate segregation of chromosomes during cell division.

k) What are inhibitory proteins that control CDK activity?

Cyclin-dependent kinases (CDKs) are a group of enzymes that play a central role in controlling the progression of the cell cycle. These CDKs must be activated at the correct stage of the cell cycle and this activation depends on their association with cyclins. However, to prevent uncontrolled cell division and ensure genomic stability, the activity of CDKs must also be properly regulated. One of the most important regulatory mechanisms involves CDK inhibitory proteins, which bind to CDKs or cyclin-CDK complexes and block their activity. These inhibitors act like "brakes" in the cell cycle machinery and are mainly divided into two major families: INK4 family and Cip/Kip family.

1. INK4 Family (Inhibitors of CDK4)

The INK4 family specifically inhibits CDK4 and CDK6, which are required for the G1 to S phase transition. These inhibitors prevent the binding of cyclin D to CDK4/6 and thereby stop the activation of the kinase.

Members of INK4 family include:

• p15 (INK4b)

• p16 (INK4a)

• p18 (INK4c)

• p19 (INK4d)

These proteins play a major role in early G1 phase and are especially important in stopping the cell cycle when there is stress or DNA damage.

2. Cip/Kip Family (CDK Interacting Protein/Kinase Inhibitory Protein)

This family is more versatile and can inhibit a wider range of CDKs, especially CDK2, CDK1 and CDK4/6. They work by binding directly to cyclin-CDK complexes and inhibiting their kinase activity.

Major members of Cip/Kip family include:

• p21 (Cip1/Waf1): Induced by p53 in response to DNA damage. It stops CDK2 and CDK1, thereby halting the cell cycle in G1 and G2.

• p27 (Kip1): Active in contact inhibition and cellular quiescence. It prevents cell cycle entry by blocking CDK2 activity.

• p57 (Kip2): Plays a key role in development and is important in tissues like placenta and nervous system.

l) What is the function of the p53 tumour suppressor gene?

The p53 gene is one of the most important tumour suppressor genes in humans. It encodes a transcription factor called p53 protein, which is often referred to as the "guardian of the genome" because of its central role in protecting cells from turning cancerous. This gene is highly conserved and its function is critical for maintaining genomic stability.

Main Function of p53

The primary function of p53 is to monitor the integrity of the DNA in the cell. When DNA damage or other cellular stress occurs (like hypoxia or oncogene activation), p53 gets activated and performs the following key functions:

1. DNA Damage Response and Cell Cycle Arrest

• One of the first actions of p53 is to stop the cell cycle. It does this by activating the transcription of p21 (Cip1), a CDK inhibitor. p21 blocks the activity of cyclin-CDK complexes, especially CDK2, which prevents the cell from entering S phase. This pause in the cycle allows time for the cell to repair its damaged DNA.

2. DNA Repair Activation

• p53 helps activate genes involved in DNA repair. If the damage is repairable, p53 allows time for these mechanisms to fix the problem. Once repaired, the cell cycle continues.

3. Apoptosis (Programmed Cell Death)

• If the DNA damage is too severe and cannot be repaired, p53 activates genes like Bax, PUMA, and NOXA which lead to apoptosis. This ensures that damaged cells do not survive and multiply, which could lead to cancer.

4. Senescence

• In some cases, instead of apoptosis, p53 causes permanent arrest of the cell cycle called senescence. These cells no longer divide and do not become cancerous.

5. Inhibition of Angiogenesis

• p53 can also inhibit blood vessel formation (angiogenesis) by repressing pro-angiogenic genes, reducing tumour growth potential.

m) How is the p53 protein activated?

The p53 protein is a tumour suppressor that remains inactive under normal cell conditions. Its activation is carefully regulated and occurs only when the cell experiences stress, especially DNA damage. This activation helps the cell decide whether to pause the cycle for repair or undergo apoptosis. The process of activation mainly depends on blocking its negative regulator and allowing p53 to become stable and active.

Mechanism of p53 Activation

The activation of p53 happens mainly in response to DNA damage, oxidative stress, hypoxia, or oncogene activation. The steps involved are:

Step 1: Detection of DNA Damage

• When DNA is damaged due to UV rays, radiation, chemicals, or errors during replication, special sensor proteins such as ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related) are activated. These proteins sense the damage and initiate a signalling cascade.

Step 2: Activation of Checkpoint Kinases

• ATM and ATR then activate downstream proteins like Chk1 and Chk2 (checkpoint kinases). These kinases phosphorylate specific target proteins that regulate the cell cycle and DNA repair, especially the p53 protein.

Step 3: Phosphorylation of p53 and MDM2

• These checkpoint kinases phosphorylate p53 at specific serine residues (like Ser15 and Ser20). This phosphorylation changes the structure of p53 and prevents it from binding with MDM2, a protein that normally adds ubiquitin to p53 for degradation. MDM2 itself can also be phosphorylated, reducing its activity. As a result, p53 becomes stable and starts to accumulate in the nucleus.

Step 4: Stabilisation and Nuclear Accumulation of p53

• Now free from MDM2 control, p53 protein accumulates in the nucleus in its active form. It becomes a transcription factor, which means it can now bind to DNA and start the expression of specific genes.

Step 5: Transcription of Target Genes

• The activated p53 turns on many important genes such as:
• p21 (Cip1): Stops the cell cycle by inhibiting CDKs

• GADD45: Involved in DNA repair

• BAX, PUMA, NOXA: Involved in apoptosis if damage is not repairable

n) How does p53 halt the cell cycle?

The p53 protein is a powerful tumour suppressor and transcription factor. It plays a very important role in protecting the cell from uncontrolled division. Whenever the cell experiences DNA damage, hypoxia, or stress signals, the p53 protein gets activated. However, it does not directly stop the cell cycle machinery. Instead, it halts the cell cycle indirectly by activating other genes that code for CDK inhibitors, especially p21 (Cip1/Waf1). This action ensures that the damaged cell does not move forward in the cell cycle until the issue is fixed.

Mechanism of Cell Cycle Arrest by p53

The mechanism through which p53 halts the cell cycle involves several key steps, which are explained below:

1. DNA Damage Sensing and p53 Activation

• When DNA damage occurs, sensor proteins like ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related) detect the problem. These proteins activate Chk1 and Chk2 kinases, which then phosphorylate and stabilize p53. Under normal conditions, p53 is unstable because it is continuously degraded by MDM2, but upon phosphorylation, p53 becomes stable and active.

2. Transcriptional Activation of p21

• Once stabilized, p53 works as a transcription factor. It enters the nucleus and binds to the promoter region of the CDKN1A gene, which encodes p21. This causes high production of p21 protein in the cell.

3. Role of p21 in Halting the Cell Cycle

• p21 protein belongs to the Cip/Kip family of CDK inhibitors. It directly binds to Cyclin-CDK complexes, especially Cyclin E-CDK2 and Cyclin A-CDK2, and inhibits their kinase activity. These complexes are necessary for G1 to S phase progression. When they are inhibited, the cell cannot proceed to DNA replication.

4. G1 Arrest (and Sometimes G2 Arrest)

• In most cases, p53 causes G1 phase arrest by stopping Cyclin E-CDK2. In some conditions, it may also help in G2 arrest by inhibiting Cyclin B-CDK1 through other mediators like 14-3-3σ. This gives time for DNA repair and avoids the passing of mutations to daughter cells.

o) What happens if DNA damage is irreparable?

When a cell experiences DNA damage, it usually activates DNA repair mechanisms to fix the damage and continue the normal cell cycle. However, if the DNA damage is too severe or irreparable, meaning it cannot be fixed by any known cellular repair systems, then the cell initiates irreversible fail-safe responses. These responses are extremely important to protect the organism from mutations that could lead to cancer or developmental defects. There are two major irreversible outcomes in such situations:

1. Apoptosis (Programmed Cell Death)

This is the most common and well-studied response when DNA damage is beyond repair. The cell activates a self-destruction program called apoptosis, which is highly regulated and highly efficient. This process is mainly controlled by the p53 tumour suppressor protein, which acts like a cellular guardian.

When p53 remains stabilised due to irreparable damage, it induces the transcription of pro-apoptotic genes like BAX, PUMA and NOXA. These proteins target the mitochondrial membrane, leading to the release of cytochrome c into the cytoplasm. Cytochrome c then binds with Apaf-1 and forms the apoptosome, which activates caspase-9 and subsequently caspase-3, initiating a cascade of proteolytic reactions that lead to DNA fragmentation, cell shrinkage, membrane blebbing and finally complete destruction of the cell.

This entire process ensures that the damaged and dangerous cell is safely removed without causing inflammation or damage to surrounding tissues.

2. Cellular Senescence (Permanent Arrest of the Cell Cycle)

If apoptosis is not triggered or in some cases like ageing tissues, the cell may instead enter a state called senescence. This is a condition where the cell remains alive and metabolically active but permanently exits the cell cycle. It can no longer divide or replicate and stays arrested in a G₀ phase-like state.

Senescence is also mainly controlled by p53, and RB (retinoblastoma protein) pathways. These proteins inhibit CDK activity and stop the progression of the cell cycle at the G1 phase. Senescent cells are often seen to accumulate in ageing tissues and may also secrete inflammatory signals {called SASP (Senescence-Associated Secretory Phenotype)}.

Although the cell does not die, it is permanently prevented from passing the damaged DNA to daughter cells. This is another protective mechanism against tumour formation.

p) How does p53 contribute to apoptosis?

The p53 protein is a tumour suppressor gene product that helps maintain the health of a cell. It does this by sensing DNA damage or other cell stress. When the damage inside the cell is too much and cannot be repaired, p53 contributes to the process of apoptosis, which means programmed cell death. This way, it helps remove damaged cells so they do not become cancerous.

How p53 Contributes to Apoptosis

It mainly works in three ways:

1. It Increases the Production of Pro-apoptotic Proteins

When p53 is activated, it goes into the nucleus and starts the production (transcription) of special genes that promote cell death. These include:

• BAX: It helps in making holes in the mitochondrial membrane, which is an important step in cell death.

• PUMA and NOXA: These are small proteins that support BAX and stop the proteins that try to save the cell.

All these proteins act together and start the mitochondrial (intrinsic) pathway of apoptosis.

2. It Helps Release Cytochrome c from Mitochondria

Because of BAX and PUMA, the outer membrane of mitochondria becomes leaky. From there, a protein called cytochrome c comes out. This cytochrome c joins with Apaf-1 and forms a structure called the apoptosome.

This structure activates caspase enzymes (like caspase-9 and caspase-3), which are responsible for breaking down the cell slowly and safely.

3. It Blocks the Proteins That Prevent Apoptosis

p53 also stops the proteins that normally try to save the cell, such as:

• Bcl-2

• Survivin

By blocking them, p53 makes sure that the cell moves forward towards apoptosis if the damage is too much.

q) What is the role of the p53 tumour suppressor gene in preventing cancer?

The p53 gene is a very important tumour suppressor gene found in almost every cell of the body. It produces a protein called p53 protein, which is also known as the "guardian of the genome". Its main job is to keep the cell healthy by checking the condition of the DNA. If any damage or mutation is found, p53 takes action to stop that cell from becoming a cancer cell.

How p53 Prevents Cancer

p53 protects the body from cancer in many ways. Here are its main roles:

1. DNA Damage Detection

• p53 is always monitoring the DNA. When it finds any damage or mutation, it quickly becomes active. This is the first step. If such damage is ignored, the cell may continue dividing with mistakes, which can lead to cancer. So, p53 first detects problems and prepares the cell to respond.

2. Halting the Cell Cycle

• Once damage is found, p53 stops the cell cycle to give the cell enough time to repair the DNA. It does this by increasing the production of a protein called p21, which blocks cyclin-CDK complexes. This pause mostly happens at the G1 checkpoint, but it can also occur at the G2 checkpoint. By stopping the cycle, p53 prevents the damaged DNA from being passed on during cell division.

3. Repairing Damaged DNA

• During the pause, p53 helps the cell to repair its damaged DNA. It activates several repair genes. If the repair is successful, the cell is allowed to continue its normal cycle. This step is very important to stop mutations from becoming permanent.

4. Triggering Apoptosis (Programmed Cell Death)

• If the DNA damage is too much and cannot be fixed, p53 activates the apoptosis pathway. This means the cell is safely destroyed before it can cause harm. p53 activates pro-apoptotic genes like BAX, PUM and NOXA. This step is very important in preventing damaged cells from turning into cancer.

5. Preventing Angiogenesis and Metastasis

• p53 prevents cancer progression by blocking signals that promote angiogenesis, which is the formation of new blood vessels needed to supply tumours. It also inhibits metastasis, stopping cancer cells from spreading to other parts of the body. This helps in restricting tumour size and maintaining localised growth.

TERMINAL QUESTIONS

1. What are the events involved in the S-phase of Interphase?

The S-phase (Synthesis phase) is a critical part of interphase in the cell cycle, occurring between the G1 (gap 1) phase and the G2 (gap 2) phase. During this phase, the cell duplicates its DNA to prepare for cell division, ensuring that the genetic material is accurately passed on to the daughter cells. This phase is essential for maintaining genomic integrity and stability.

Key Events in the S-phase

1. DNA Replication:

The main event of the S-phase is DNA replication. This process ensures that the entire genome is copied so that each daughter cell will have an identical set of chromosomes. The helicase enzyme unwinds the DNA double helix, creating two single strands. These single strands act as templates for the synthesis of new complementary strands.

2. Activation of DNA Polymerases:

DNA polymerases are key enzymes that catalyze the addition of new nucleotides to the growing strand. On the leading strand, DNA polymerase synthesizes continuously in the 5' to 3' direction. On the lagging strand, DNA is synthesized in small segments known as Okazaki fragments, which are later joined together by DNA ligase to form a continuous strand.

3. Formation of Replication Forks:

The points where the DNA double helix begins to unwind are called replication forks. These forks are the sites where DNA replication occurs and they form at multiple origins of replication along the DNA molecule. The replication forks move in both directions as the DNA is unwound and replicated.

4. Chromatin Remodeling:

As the DNA is replicated, the chromatin undergoes structural changes. The chromatin, which is tightly packed in the nucleus, is temporarily loosened by chromatin remodeling complexes to allow access to the DNA by the replication machinery. After replication, the chromatin is reassembled to restore its compact structure.

5. Centrosome Duplication:

During the S-phase, centrosomes, which are key structures involved in organizing the microtubules of the mitotic spindle, duplicate. The centrosomes ensure that, during mitosis, the chromosomes are accurately separated between the two daughter cells. The process of centrosome duplication is coordinated with DNA replication.

6. Checkpoint Regulation:

The S-phase is carefully monitored by the cell cycle checkpoints to ensure that DNA replication occurs without errors. If any damage or replication stress is detected, the cell cycle is paused to allow for repair. Proteins like ATM and ATR are involved in sensing DNA damage and activating repair pathways. If the damage cannot be repaired, the cell may trigger apoptosis (programmed cell death) to prevent passing on mutations.

2. Give a brief note on the control of the Cell Cycle.

The cell cycle is a highly regulated process that ensures cells grow, replicate their DNA and divide accurately. Its control is crucial for normal development, tissue repair and prevention of diseases like cancer. This control is achieved mainly through a combination of regulatory proteins, checkpoints, inhibitory pathways and external signaling factors, all of which coordinate to monitor and regulate the progression of the cycle at every stage.

1. Cyclins and CDKs – The Core Regulators

Cyclin-dependent kinases (CDKs) are special enzymes that become active only when they bind to a protein called cyclin, to form a cyclin-CDK complex. Different cyclins appear and disappear at specific times in the cell cycle, and this timing controls CDK activity. Each cyclin-CDK complex triggers important events of a particular phase of cell cycle.

• In the G1 phase, Cyclin D binds to CDK4 or CDK6 to push the cell toward the S phase.

• During the S phase, Cyclin E and later Cyclin A bind to CDK2 to promote DNA synthesis.

• In the G2 and M phases, Cyclin B joins with CDK1 to drive the cell into mitosis.

These complexes phosphorylate (add phosphate) to key proteins, switching them on or off to move the cycle forward.

2. Cell Cycle Checkpoints – Surveillance System

Checkpoints act like security checks. They make sure everything is correct before the cycle continues.

• G1/S checkpoint: Checks for DNA damage and ensures the cell is ready for replication. If damaged DNA is detected, the cycle is paused.

• G2/M checkpoint: Ensures DNA has been copied completely and correctly before entering mitosis.

• Spindle checkpoint: During metaphase, it confirms that all chromosomes are attached to spindle fibres properly.

If problems are found, the cycle stops temporarily so the cell can repair the issue. If not repairable, the cell may self-destruct (apoptosis).

3. Inhibitors and Tumour Suppressor Genes

Proteins like p21, p27 and p16 act as natural brakes. They stop CDKs from working when the cell needs time to fix errors. The p53 tumour suppressor gene is very important here. When DNA damage occurs, p53 becomes active and tells the cell to produce p21, which then blocks CDK activity, halting the cycle in G1 phase. This gives time for repair or leads to apoptosis if the damage is serious.

4. External Signals and Growth Factors

Cells also respond to signals from outside. Growth factors like epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) encourage the cell to start the cycle by increasing cyclin levels. On the other hand, signals like TGF-beta promote CDK inhibitors to stop unwanted growth.

3. What is Cyclin-CDKs kinases? Write a brief note on the relation of cyclin with CDKs.

Cyclin-CDK kinases are enzyme complexes that control the progression of the eukaryotic cell cycle. These complexes consist of two main components: a cyclin protein and a cyclin-dependent kinase (CDK). CDKs are serine/threonine protein kinases that are present in the cell in an inactive form. They require the binding of a regulatory protein, called a cyclin, to become active. Once a cyclin binds to a CDK, the complex becomes enzymatically active and can phosphorylate various target proteins involved in controlling key steps of the cell cycle, such as DNA replication, chromosome condensation and mitotic spindle formation.

The activity of cyclin-CDK complexes is regulated at multiple levels, including cyclin synthesis and degradation, phosphorylation and dephosphorylation of CDKs, and the presence of CDK inhibitors (CKIs). This regulation ensures that each phase of the cell cycle occurs only once and in the proper order, preventing genomic instability or uncontrolled cell division. Cyclin-CDK kinases play a key role in maintaining genome integrity and are considered essential guardians of normal cell proliferation.

Relationship of Cyclins with CDKs

The relationship between cyclins and CDKs is highly specific and tightly regulated. Cyclins are named so because their levels rise and fall cyclically during the cell cycle. Each phase of the cell cycle is controlled by a specific cyclin-CDK pair. The main types of relationships between cyclins and CDKs can be explained in the following steps:

1. Cyclins Activate CDKs:

CDKs are inactive on their own. They require a specific cyclin to bind and cause a conformational change that activates their kinase activity. Without cyclins, CDKs cannot function.

2. Stage-Specific Regulation:

Different cyclins are synthesized and degraded at different stages of the cell cycle, and each binds to specific CDKs to control that phase. For example:

• Cyclin D-CDK4/6 regulates the early G1 phase.

• Cyclin E-CDK2 controls the transition from G1 to S phase.

• Cyclin A-CDK2 is active during S phase to support DNA replication.

• Cyclin B-CDK1 is required for the G2 to M phase transition and mitosis initiation.

3. Sequential Activation:

The binding of cyclins to CDKs occurs in a sequential manner. One cyclin-CDK complex activates processes that lead to the formation or activation of the next complex. This ensures orderly progression through the cell cycle.

4. Destruction of Cyclins:

Once their function is complete, cyclins are degraded by the ubiquitin-proteasome pathway. This inactivates the CDK and stops that phase, preventing re-entry until the next proper signal arrives.

4. How does the p53 tumour suppressor gene regulate the cell cycle?

The p53 tumor suppressor gene plays a critical role in safeguarding the integrity of the genome by controlling the cell cycle. It is often referred to as the "guardian of the genome" due to its essential function in preventing the propagation of damaged or mutated DNA, which is a key factor in cancer development. p53's regulation of the cell cycle is mainly focused on halting the cycle in response to DNA damage, thereby allowing time for repair or inducing apoptosis if the damage is irreparable. This process helps prevent the accumulation of mutations that could lead to tumor formation.

There are five key steps involved in how p53 regulates the cell cycle:

1. Detection of DNA Damage and Activation of p53

When a cell experiences DNA damage due to radiation, toxins, oxidative stress, or other reasons, certain kinases like ATM and ATR are activated. These kinases phosphorylate the p53 protein. Normally, p53 is degraded quickly by MDM2, but phosphorylation prevents this degradation, causing p53 to accumulate in the nucleus and become active.

2. Transcriptional Activation of p21 Gene

Once activated, p53 acts as a transcription factor. It binds to specific DNA sequences and activates the transcription of many genes. One of the most important genes it activates is CDKN1A, which produces p21 protein. p21 is a type of cyclin-dependent kinase inhibitor (CDKI).

3. Inhibition of Cyclin-CDK Complexes

p21 protein binds to Cyclin-CDK complexes, such as Cyclin E-CDK2, which are normally required for the transition from G1 phase to S phase of the cell cycle. By inhibiting these complexes, p21 stops the phosphorylation of Retinoblastoma protein (Rb), which in turn prevents the release of E2F transcription factors that are needed for DNA replication.

4. Cell Cycle Arrest at G1/S or G2/M Checkpoints

Due to the inhibition of Cyclin-CDK activity, the cell is arrested at either the G1/S or G2/M checkpoint, depending on the timing and location of the damage. This gives the cell sufficient time to repair damaged DNA. If the damage is repaired successfully, the cell cycle resumes normally.

5. Activation of Apoptosis if DNA Damage is Irreparable

If the DNA damage is beyond repair, p53 shifts its function and activates pro-apoptotic genes like BAX, PUMA, NOXA and others. These genes lead to programmed cell death (apoptosis). This ensures that severely damaged or potentially cancerous cells do not survive or divide.

Read More:

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