Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.
Background All living organisms are the products of repeated rounds of cell growth and division. During this process, known as the cell cycle, a cell duplicates its contents and then divides in two. The purpose of the cell cycle is to accurately duplicate each organism's DNA and then divide the cell and its contents evenly between the two resulting cells. In eukaryotes, the cell cycle consists of four main stages: G1, during which a cell is metabolically active and continuously grows; S phase, during which DNA replication takes place; G2, during which cell growth continues and the cell synthesizes various proteins in preparation for division; and the M (mitosis) phase, during which the duplicated chromosomes (known as the sister chromatids) separate into two daughter nuclei, and the cell divides into two daughter cells, each with a full copy of DNA. Compared to the eukaryotic cell cycle, the prokaryotic cell cycle (known as binary fission) is relatively simple and quick: the chromosome replicates from the origin of replication, a new membrane is assembled, and the cell wall forms a septum which divides the cell into two.
As the eukaryotic cell cycle is a complex process, eukaryotes have evolved a network of regulatory proteins, known as the cell cycle control system, which monitors and dictates the progression of the cell through the cell cycle. This system acts like a timer, or a clock, which sets a fixed amount of time for the cell to spend in each phase of the cell cycle, while at the same time it also responds to information received from the processes it controls. The cell cycle checkpoints play an important role in the control system by sensing defects that occur during essential processes such as DNA replication or chromosome segregation, and inducing a cell cycle arrest in response until the defects are repaired. The main mechanism of action of the cell cycle checkpoints is through the regulation of the activities of a family of protein kinases known as the cyclin-dependent kinases (CDKs), which bind to different classes of regulator proteins known as cyclins, with specific cyclin-CDK complexes being formed and activated at different phases of the cell cycle. Those complexes, in turn, activate different downstream targets to promote or prevent cell cycle progression.
G1 (restriction) checkpoint
The G1 checkpoint, also known as the restriction point in mammalian cells and the start point in yeast, is the point at which the cell becomes committed to entering the cell cycle. As the cell progresses through G1, depending on internal and external conditions, it can either delay G1, enter a quiescent state known as G0, or proceed past the restriction point. DNA damage is the main indication for a cell to "restrict" and not enter the cell cycle. The decision to commit to a new round of cell division occurs when the cell activates cyclin-CDK-dependent transcription which promotes entry into S phase. This check point ensures the further process.
During early G1, there are three transcriptional repressors, known as pocket proteins, that bind to E2F transcription factors. The E2F gene family is a group of transcription factors that target many genes that are important for control of the cell cycle, including cyclins, CDKs, checkpoint regulators, and DNA repair proteins. Misregulation of the E2F family is often found in cancer cases, providing evidence that the E2F family is essential for the tight regulation of DNA replication and division. The three pocket proteins are Retinoblastoma (Rb), p107, and p130, which bind to the E2F transcription factors to prevent progression past the G1 checkpoint.
The E2F gene family contains some proteins with activator mechanisms and some proteins with repressing mechanisms. P107 and p130 act as co-repressors for E2F 4 and E2F 5, which work to repress transcription of G1-to-S promoting factors. The third pocket protein, Rb, binds to and represses E2F 1, E2F 2, and E2F 3, which are the E2F proteins with activating abilities.
Positive feedback plays an essential role in regulating the progression from G1 to S phase, particularly involving the phosphorylation of Rb by a Cyclin/CDK protein complex. Rb without a phosphate, or unphosphorylated Rb, regulates G0 cell cycle exit and differentiation. During the beginning of the G1 phase, growth factors and DNA damage signal for the rise of cyclin D levels, which then binds to Cdk4 and Cdk6 to form the CyclinD:Cdk4/6 complex. This complex is known to inactivate Rb by phosphorylation. However, the details of Rb phosphorylation are quite complex and specific compared to previous knowledge about the G1checkpoint. CyclinD:Cdk4/6 places only one phosphate, or monophosphorylates, Rb at one of its fourteen accessible and unique phosphorylation sites. Each of the fourteen specific mono-phosphorylated isoforms has a differential binding preference to E2F family members, which likely adds to the diversity of cellular processes within the mammalian body.
E2F 4 and E2F 5 are dependent on p107 and p130 to maintain their nuclear localization. However, Cyclin D:Cdk 4/6 also phosphorylates p107 and p130, a process which releases their bind from E2F 4 and 5 (which then escape to the cytoplasm), and allowing for E2F 1–3 to bind to the DNA and initiate transcription of Cyclin E. Rb proteins maintain their mono-phosphorylated state during early G1 phase, while Cyclin E is accumulating and binding to Cdk2.
CyclinE:Cdk2 plays an additional important phosphorylation role in the G1-to-S transition. Particularly, CyclinE:Cdk2 promotes a positive feedback loop which creates an "all or nothing" switch. In many genetic control networks, positive feedback ensures that cells do not slip back and forth between cell cycle phases Cyclin E:Cdk2 proceeds to phosphorylate Rb at all of its phosphorylation sites, also termed "hyper-phosphorylate", which ensures complete inactivation of Rb. The hyper phosphorylation of Rb is considered the late G1 restriction point, after which the cell cannot go backwards in the cell cycle. At this point, E2F 1-3 proteins bind to DNA and transcribe Cyclin A and Cdc 6.
Cyclin-dependent kinase inhibitor 1B (CDKN1B), also known as p27, binds to and prevents the activation of CyclinE:Cdk2 by inhibition. However, as Cyclin A accumulates and binds to Cdk2, they form a complex and inhibit p27. The G1 phase cyclin-dependent kinase works together with S phase cyclin-dependent kinase targeting p27 for degradation. In turn, this allows for full activation of Cyclin A:Cdk2, a complex which phosphorylates E2F 1-3 initiating their disassociation from the DNA promoter sites. This allows E2F 6–8 to bind to the DNA and inhibit transcription. The negative feedback loop used to successfully inhibit the inhibitor, p27, is another essential process used by cells to ensure mono-directional movement and no backtrack through the cell cycle.
When DNA damage occurs, or when the cell detects any defects which necessitate it to delay or halt the cell cycle in G1, arrest occurs through several mechanisms. The rapid response involves phosphorylation events that initiate with either kinase ATM (Ataxia telangiectasia mutated) or ATR (Ataxia Telangiectasia and Rad3 related), which act as sensors, depending on the type of damage. These kinases phosphorylate and activate the effector kinases Chk2 and Chk1, respectively, which in turn phosphorylate the phosphatase Cdc25A, thus marking it for ubiquitination and degradation. As Cdc25A activates the previously mentioned cyclin E-CDK2 complex by removing inhibitory phosphates from CDK2, in the absence of Cdc25A, cyclin E-CDK2 remains inactive, and the cell remains in G1.
To maintain the arrest, another response is initiated, by which Chk2 or Chk1 phosphorylate p53, a tumor suppressor, and this stabilizes p53 by preventing it from binding Mdm2, a ubiquitin ligase which inhibits p53 by targeting it for degradation. The stable p53 then acts a transcriptional activator of several target genes, including p21, an inhibitor of the G1-to-S promoting complex cyclin E-CDK2. In addition, another mechanism by which p21 is activated is through the accumulation of p16 in response to DNA damage. p16 disrupts cyclin D-CDK4 complexes, thus causing the release of p21 from the complexes, which leads to the dephosphorylation and activation of Rb, which allows Rb to bind and inhibit E2F 1–3, thus keeping the cell from transitioning to S phase. Recently, some aspects of this model have been disputed.
G2 checkpoint
Following DNA replication in S phase, the cell undergoes a growth phase known as G2. During this time, necessary mitotic proteins are produced and the cell is once more subjected to regulatory mechanisms to ensure proper status for entry into the proliferative Mitotic (M) phase. Multiple mechanistic checkpoints are involved in this transition from G2 to M, with a common uniting factor of cyclin-Cdk activity.
Although variations in requisite cyclin-Cdk complexes exist across organisms, the necessity of the kinase activity is conserved and typically focuses on a single pairing. In fission yeast three different forms of mitotic cyclin exist, and six in budding yeast, yet the primary cyclin utilized is cyclin B. Cyclin B will serve as reference for discussion of the G2/M checkpoint transition.
Similar to S Phase, G2 experiences a DNA damage checkpoint. The cell is once more examined for sites of DNA damage or incomplete replication, and the kinases ATR and ATM are recruited to damage sites. Activation of Chk1 and Chk2 also transpire, as well as p53 activation, to induce cell cycle arrest and halt progression into mitosis. An additional component of S phase, the Pre-Replicative Complex, must be inactivated via cyclin B-Cdk1 phosphorylation.