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The Eukaryotic Cell Cycle Checkpoints Biology Essay

Introduction

The cell cycle is a fundamental event evolved in all living organisms. In fact it is substantially important to be considered as a core feature of a viable cell. The cell cycle is the process by which a common ancestor generated the diversity of organisms found today.

In single cell organisms, including many eukaryotes, one complete cell cycle generates a new individual. However in multicellular eukaryotes, several of these cycles give rise to a huge number of cells and the group of all these cells make up the organism.

The cell cycle is a series of events that result in 2 daughter progeny from 1 parent cell. It involves four different stages, G1-Phase; S-Phase; G2-Phase; and M-Phase, and they occur in the same order (Fig 2) [11]. The most important stages of the cell cycle are S-Phase, during which DNA replication takes place, and M-Phase, when replicated chromosomes are separated [1]. The M and S-Phase are separated by distinct intervals call G1 (between M-S Phase) and G2 (between S-M Phase). The crucial role of the cell cycle checkpoints is to monitor and govern the efficiency of these processes. Failure of this mechanism can lead to abnormal cell proliferation and potentially cancer. For example, if either of the M or S-Phase is repeated then it can result in half DNA content (M-Phase) or double DNA content/ polyploidy (S-Phase), both lethal [4]. Therefore a series of checkpoints are involved at specific stages of the cell cycle to maintain the order of events. Most of these checkpoints are conserves within all eukaryotes [3].

The regulation of different CDK/cyclin complexes is responsible for well-organised cell cycle progression. CDKs are present at constant level throughout the cell cycle; however it is the fluctuation of cyclin concentration that drives this regulation (Fig 3).

G1-Phase

This interval in the cell cycle could last up to During G1 the cell cycle gathers biological machinery like enzymes and other molecules, required for DNA replication [2]. Synthesis of new organelles, enzymes such as DNA polymerase, Gyrase and Helicase and other structural proteins occur during this interval, to prepare the cell for S-Phase (Fig 1). The cell waits for growth factor signals, and investigates whether there is sufficient energy and space before initiating these processes. A ‘START’ restriction point does operate during this phase; this is predominant in S. cerevisiae. It prevents the cell from progressing to S-Phase until appropriate signals, induced by exterior factors, are detected. For example D cyclins are produced, depending upon the growth factor signal, which associate with CDK2, CDK3, CDK4 and CDK6. It is important for the cell to certify the presence of relevant factors, which help the cell advance to S-Phase, as once the cell commits to proceed past this restriction point, it cannot ‘undo’ this decision [4].

The CDC28 CKD can associate with CLN cyclins, CLN1; CLN2; or CLN3. It was found that when the concentration of CDC28-CLN3 complex reaches a certain level, SBF and MBF transcription factors (TFs) are activated by the kinase. The SBF promotes transcription of G1 cyclin CLN1 and CLN2. These newly synthesised cyclins associate with CDC28 to activate mitotic and S-Phase CLB cyclins, thus progressing through the cell cycle [4].

Another checkpoint mechanism dwells within the G1-Phase. E2F is a TF that promotes expression of essential genes required for DNA synthesis. The proteins p107 and p130 bind to E2F and inactivate it. The G1 cyclins – CYCLIN D-CDK4/CDK6 and CYCLIN E-CDK2 phosphorylate p107 and p130, which leads to the release of E2F and progression from G1 to S-Phase. Another protein responsible for inactivating E2F is Rb-1, by binding to E2F in its unphosphorylated form (Fig 6). The Rb-1 also induces repression of the cell cycle by promoting chromatin remodelling. The CDK-cyclinD complex phosphorylates Rb-1 to oppose this repression. The CDK-cyclinD complex also induces cyclin E synthesis, which is required in the S-Phase [8].

Animal cells often withdraw from the cell cycle and enter G0-Phase, either due to exterior – growth and division factors are repressed or because of their differentiated condition, e.g. red blood cells.

S-Phase

The duration of the S-Phase is about 6 hours. It lasts so long because the whole genome is to be replicated in this period. DNA replication requires 2 DNA polymerases DNA Pol A(α) and Pol D(δ) [10]. Pol A synthesises primers and Pol D extends it, within a replication fork. In eukaryotes there are several origins of replication so many replication forks are formed simultaneously. Along with DNA synthesis, the cell also has to produce enough proteins (histones) to shield the new DNA.

Cyclin E is expressed late in G1-Phase and early S1-Phase, and forms a complex with CDK2. Cyclin E assists in DNA synthesis by phosphorylating components of the DNA| replication machinery. Cyclin A is capable of activating two CDKs, and is present during S-Phase and M-Phase. It accumulates during S-Phase and is quickly degraded before metaphase (M-Phase) [9]. Initiation of DNA replication requires participation of CDK-cyclinA and CDK-cyclinE complexes, however the function of these complexes in the initiation is now known. Cyclin E induce DNA replication by associating with Cdc6, which regulates initiation of DNA replication. Cyclin A has 2 functions, it promotes DNA synthesis by the replication complexes already assembled, and second it inhibits the assembly of new complexes. This allows cyclin E to promote replication, whereas cyclin A prevents assemble of new components. This characteristic of cyclin A certifies that G1 ends before S-Phase commences, preventing re-initiation of replication complexes until the next cycle [3].

G2-Phase

The G2-Phase is the interval between S-Phase and M-Phase. The cell growth continues during this stage and proteins are synthesised in preparation for mitosis (M-Phase).

In yeast, S. pombe, CDC28 & Cdc2 CDKs are phosphorylated on 2 residues, Tyr15 and Thr161. Phosphorylation of Thr161 activates the kinase property of the CDK whereas phosphorylation of Tyr15 inhibits this function. The phosphorylation of Tyr15, dominates the CDK activity regardless of the phosphorylation state of Thr161, and is carried out by Wee1. This process is opposed by Cdc25 phosphatase, which removes phosphate groups from Tyr15. It is thought that regulation of Wee1 and Cdc25 by external factors and signals, forms basis of G2-M checkpoint. This checkpoint in mammalian cells is more complex and involves another key residue in the mammalian CDK, which is a homologue of the yeast CDK. The key residue is Thr14 and phosphorylation of the CDK by Myt1 inhibits kinase activity [4].

If the DNA is damages during replication, it is important for the cell to respond to this crisis. DNA damage results in activation of ATM and ATR kinases. This causes a kinase cascade, Chk1 kinase is phosphorylated by these kinases, Chk1 phosphorylates Cdc25 phosphatase and inactivates it. This prevents dephosphorylation of cyclin B1-Cdk1 at Thr14 and Tyr15. Cyclin B1-Cdk1 activation requires dephosphorylation at these residues by Cdc25, along with phosphorylation of Thr161 by a cyclin activating kinase (CAK). Mitotic initiation requires activated Cdk1, without this the cell will be arrested in G2 transition [5].

In addition to this ATM/ATR can directly or through phosphorylation-dependent activation of Chk2, phosphorylate p53, which leads to increased expression of p21CIP1, GADD45, and 14-3-3σ. These proteins help the cell maintain G2 arrest transition. p21CIP1 interacts with cyclin B1-Cdk1, and prevent the phosphorylation process of Cdk1 (Thr161) by CAK. GADD45 binds to Chk1 subunit, which results in dissociation of Cyclin B1-Cdk1 complex. 14-3-3σ is thought to restrict the transportation of Cyclin B1-Cdk1 complex into the nucleus. Activities of these proteins forbid progression of the cycle and the cells halt in G2-Phase. Apart from DNA damage, absence of factors such as mitogen can also influence cyclin B1-Cdk1 activity and impose a G2 arrest [5].

M-Phase

During this stage chromosome distribution and cell division takes place to produce 2 identical daughter cells [11]. The M-Phase itself contains distinct series of segregation events. These are Prophase, Pro-Metaphase, Metaphase, Anaphase, Telophase and Cytokinesis [13].

In Prophase chromosome are condensed and therefore become visible under light microscope. The sister chromatids are associated together and the mitotic spindle formation is initiated.

Nuclear membrane disintegrates in Pro-Metaphase (Fig 4), which allows chromosomes to attach to spindle fibres. When all the chromosomes are attached to the spindle, anaphase-promoting complex (APC) is activated by Cdc20/Spl1

During Metaphase the chromosomes align at the equator of the spindle. Cyclin A is degraded to prevent the cell from repeating M-Phase before the next cycle [3].

There is a checkpoint which governs the assembly of chromosomes along the spindle equator. The function of this mitotic checkpoint is to prevent pre-mature mitotic exit. At metaphase, progression to anaphase is only possible after activation of APC. This complex promotes degradation of proteins that hold the replicated chromosomes together or proteins that keep the cell in mitosis. As the APC is activated by Cdc20, if the cell is deprived of Cdc20, the cell arrests in mitosis [12].

After the progression to anaphase, the sister chromatids dissociate and simultaneously move apart, because of separation of spindle poles due to repulsion of microtubules (identical charge at ends). Cyclin B is also degraded, consequently inactivating the M-Phase CKD. This again prevents the cell from re-entering M-Phase. The machinery that degrades Cyclin B also targets the protein that binds and holds sister chromatids together, assisting in the segregation of genome.

During Telophase, the spindle fibres continue to shorten and the daughter chromosomes (sister chromatids) reach the opposite poles of the cell. This process is followed by cytokinesis, cell division. Actin and myosin filaments form a contractile ring, around the interior membrane of the cell, along the metaphase equator line (Fig 5).

Summary

The cell cycle is a process that is conserved throughout life. It involves a number of checkpoints which make sure that the cycle progresses in an appropriate order. Possession of such surveillance machinery is a major benefit to the cell and enhances the chances of survival to a certain extent. The function of a checkpoint is to certify that all processes in the earlier phase are accurately completed and arrest the cell if this condition is not met. Progression of the cell cycle requires exterior and interior signals and growth factors. The G1 restriction point is probably the most important checkpoint as once the cell commits to proceed; it cannot halt in G1 until the cycle is completed.

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