G2-M DNA damage checkpoint

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Steps of the cell cycle. The G2-M checkpoint occurs between the G2 and M phases. Cell Cycle 2-2.svg
Steps of the cell cycle. The G2-M checkpoint occurs between the G2 and M phases.
G2-M arrest G2-Marrest.png
G2-M arrest

The G2-M DNA damage checkpoint is an important cell cycle checkpoint in eukaryotic organisms that ensures that cells don't initiate mitosis until damaged or incompletely replicated DNA is sufficiently repaired. Cells with a defective G2-M checkpoint will undergo apoptosis or death after cell division if they enter the M phase before repairing their DNA. [1] The defining biochemical feature of this checkpoint is the activation of M-phase cyclin-CDK complexes, which phosphorylate proteins that promote spindle assembly and bring the cell to metaphase. [2]

Contents

Cyclin B-CDK 1 activity

CyclinB-Cdk1 Hysteresis Graph CyclinB-Cdk1 Hysteresis Graph.svg
CyclinB-Cdk1 Hysteresis Graph

The cell cycle is driven by proteins called cyclin dependent kinases that associate with cyclin regulatory proteins at different checkpoints of the cell cycle. Different phases of the cell cycle experience activation and/or deactivation of specific cyclin-CDK complexes.

CyclinB-CDK1 activity is specific to the G2/M checkpoint. Accumulation of cyclin B increases the activity of the cyclin dependent kinase Cdk1 human homolog Cdc2 as cells prepare to enter mitosis. Cdc2 activity is further regulated by phosphorylation/dephosphorylation of its corresponding activators and inhibitors. Through a positive feedback loop, CyclinB-Cdc2 activates the phosphatase Cdc25 which in turn deactivates the CyclinB-Cdc2 inhibitors, Wee1 and Myt1. Cdc25 activates the complex through the removal of phosphates from the active site while Wee1 inactivates the complex through the phosphorylation of tyrosine residues, specifically tyrosine-15. [3]

This loop is further amplified indirectly through the coordinated interaction of the Aurora A kinase and the Bora cofactor. During the G2 phase, Bora accumulates and forms an activation complex with Aurora A. This complex then regulates the activation of Polo-like kinase 1 (Plk1). Plk1 phosphorylates Wee1, targeting it for degradation through the SCF ubiquitin ligase complex (SCF complex), and activates Cdc25 through phosphorylation with combined action activating Cdc2. The combined activity and complex of Cdc2, Cdc25, and Plk1 with the accumulation of cyclin B activates the CyclinB-Cdc2 complex, promoting entry into mitosis. [4]

Many proteins involved in this positive feedback loop drive the activation of the CyclinB-Cdc2 complex because entry into mitosis requires an all-or-none response. The Novak-Tyson model is a mathematical model used to explain such regulatory loop that predicted the irreversible transition into mitosis driven by hysteresis. [5] Through experiments in Xenopus laevis cell-free egg extracts, such model was confirmed as the basis for entry into mitosis. Once cyclin concentration reaches a certain minimum activation threshold, Cdc2 is rapidly activated. It remains in this state until activity falls below a separate inactivation threshold at which it is abruptly inactivated through tyrosine phosphorylation by Wee1 and Myt1. In the case of unreplicated DNA, the cyclin concentration threshold for Cdc2 activation is further increased. Through this mechanism, there exists two separate steady-state conditions separated by an unstable steady state. The bistable and hysteretic nature of CyclinB-Cdc2 ensures a highly regulated nature of the G2/M checkpoint. [6]

Pathway response to DNA damage

Proteins that localize to sites of DNA damage in the G2 phase initiate a signaling cascade that regulates important components of the pathway, as described above, therefore controlling mitotic entry via CyclinB-Cdc2 activity. Negative regulation of CyclinB-Cdc2 activity results in a delay in mitotic entry, which is important for cells to repair any DNA damage that may have accumulated after S phase and necessary before cell division can continue.

Proteins that function in the G2-M checkpoint were originally identified in yeast screens that looked for mutants which show enhanced sensitivity to radiation, termed "rad" mutants. [1] Inefficient repair of DNA damaged by ionizing radiation or chemical agents in these mutants revealed proteins essential in this pathway. Early signaling proteins in the checkpoint pathway are members of a family of phosphatidylinositol 3-kinases, rad3 in yeast and ATR in vertebrates, that are believed to localize to sites of DNA damage. [7] Rad3 phosphorylates rad26 which is required to initiate, but not maintain the checkpoint. Rad3 also phosphorylates a number of other proteins whose absence abolishes checkpoint DNA repair, including rad1, rad9, hus1 and rad17. [1] It has been hypothesized that rad9, hus1 and rad17 are similar to proteins involved in forming the clamp that increases the processivity of DNA polymerase during DNA replication. [8] In agreement with this idea, rad17 is similar to proteins involved in loading the clamp onto DNA. This supports a model where phosphorylation by rad3 causes recruitment of these proteins to sites of DNA damage where they mediate the activity of DNA polymerases involved in DNA repair. [1]

The main rad3 effector is the kinase Chk1, which is required for the G2-M arrest in response to DNA-damaging agents. [9] Chk1 is an effector protein kinase that maintains mitotic cyclin in an inactive state and is phosphorylated by rad3 between S phase and mitosis, implicating its specific role in G2 arrest. [10] Its upregulation through overexpression can induce arrest independent of DNA damage. [11] In addition, overexpression of Chk1 rescues the radiation sensitivity of rad mutants, presumably by allowing DNA repair to take place before entry into mitosis. [7]

The presence of DNA damage triggers the ATM (Ataxia telangiectasia mutated) or ATR (Ataxia Telangiectasia and Rad3 related) pathways which activate the Chk2 and Chk1 kinases, respectively. These kinases act upstream of Cdc25 and Wee1, the direct regulators of the CyclinB-Cdc2 complex. Chk1 and Chk2 phosphorylate Cdc25, inhibiting its phosphorylating activity and marking it for ubiquitinated degradation. [11] [12] These pathways also stimulate the tumor suppressor p53. p53 regulates the function of the Cdk2 inhibitor p21 and the 14-3-3 proteins that phosphorylate (and thereby inactivate) and sequester Cdc25 in the cytoplasm, respectively. [13] Recent studies have also suggested that Cdk1 and 14-3-3 positively regulate Wee1 in a similar manner. The hyperphosphorylation of Wee1 by Cdk1 allows for the binding of 14-3-3, sequestering Wee1 to the nucleus and enhancing its ability to phosphorylate Cdc2. [14] The phosphorylation of both Wee1 and Cdc25 prevents Cdc2 activation. [12]

The ATM/ATR pathway also results in the negative regulation of Plk1 that contributes to the stability of Wee1. The stabilization of Wee1 and Myt1 ensures the cells arrest in G2 and allows for DNA repair. [13] [15]

Multiple pathways are involved in the checkpoint response and thus, the targeting of Cdc25 is not the sole mechanism underlying cell cycle delay, as some models have proposed. The cooperativity between the positive regulation of Wee1 and the negative regulation of Cdc25 by Chk1 in response to unreplicated or damaged DNA results in a strong G2 arrest. [1] [11] [13] [15] The increase in the amount of Wee1 and the decrease in the amount of Cdc25 contributes to the increase in the cyclin B concentration threshold in the hysteresis loop needed to drive the cell into mitosis.

Maintaining the checkpoint

Rad3 is required for activation of Chk1 and initiation of G2 arrest, but different proteins are believed to maintain G2 arrest so that sufficient DNA repair can occur. One such protein is rad18 that is required for G2 arrest even when Chk1 is phosphorylated and active. Thus, rad18 is required for G2/M checkpoint maintenance while Chk1 is required for checkpoint initiation. [16] This is further supported by its additional function in DNA repair, specifically in the maintenance of chromosomal structures. Its necessity is demonstrated by the fact that in the absence of rad18, DNA is unable to be repaired even when G2 arrest is prolonged by other means.

The maintenance of such arrest in the G2 phase is further sustained by p53 and p21. In the absence of p53 or p21, it was demonstrated that radiated cells progressed into mitosis. [17] The absence of p21 or 14-3-3 cannot sufficiently inhibit the CyclinB-Cdc2 complex, thus exhibiting the regulatory control of p53 and p21 in the G2 checkpoint in response to DNA damage. [12] p53 mutations can result in a significant checkpoint deficit, which has important implications in the treatment of cancer.

Checkpoint inactivation

Inactivation of both Wee1 and Cdc25 abolishes the G2-M DNA damage checkpoint. Absence of Wee1 or removal of the tyrosine-15 site removes negative regulation of Cdc2 activity and causes cells to enter mitosis without completing repair, which effectively abolishes the G2-M checkpoint. [18] Absence of Cdc25 arrests cells in G2, but still allows activation of the G2-M checkpoint, implicating that both the activation of Wee1 and deactivation of Cdc25 as important regulatory steps in the checkpoint. [11]

Inactivation of Chk1 is sufficient to surpass the checkpoint and promote entry into mitosis, regardless if DNA damage is repaired. Yet, little is still known about the exact mechanism regarding checkpoint termination with possible mechanisms including protein phosphatases reversing activating phosphorylations, targeted ubiquitin degradation of activating proteins, and checkpoint antagonists promoting mitosis through independent pathways. [10]

Cancer

Many cell cycle regulators like Cdks, cyclins, and p53 have been found to have abnormal expression in cancer. More specifically, they have been implicated in being involved in the G2/M transition by localizing to the centrosome, which thus leads to studies in manipulating such proteins in order to improve cancer's sensitivity to radiation and chemotherapy. [13] Chk1 has important implications in drug targeting for cancer as its function acts in response to DNA damage. The cytotoxic effects of chemotherapy are currently being studied in the modulation of the G2/M transition, concerning both checkpoint abrogation or checkpoint arrest. [19] Many therapies focus on inactivating the checkpoint in order to force cells with excess DNA damage to proceed through mitosis and induce cell death. [12]

Related Research Articles

<span class="mw-page-title-main">Cell cycle</span> Series of events and stages that result in cell division

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that causes it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division.

Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor) is the cyclin-Cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF promotes the entrance into mitosis (the M phase) from the G2 phase by phosphorylating multiple proteins needed during mitosis. MPF is activated at the end of G2 by a phosphatase, which removes an inhibitory phosphate group added earlier.

G<sub>2</sub> phase Second growth phase in the eukaryotic cell cycle, prior to mitosis

G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.

<span class="mw-page-title-main">Cell cycle checkpoint</span> Control mechanism in the eukaryotic cell cycle

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.

<span class="mw-page-title-main">G1/S transition</span> Stage in cell cycle

The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated checkpoint can lead to cellular transformation and disease states such as cancer.

Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.

<span class="mw-page-title-main">Ataxia telangiectasia and Rad3 related</span> Protein kinase that detects DNA damage and halts cell division

Serine/threonine-protein kinase ATR, also known as ataxia telangiectasia and Rad3-related protein (ATR) or FRAP-related protein 1 (FRP1), is an enzyme that, in humans, is encoded by the ATR gene. It is a large kinase of about 301.66 kDa. ATR belongs to the phosphatidylinositol 3-kinase-related kinase protein family. ATR is activated in response to single strand breaks, and works with ATM to ensure genome integrity.

<span class="mw-page-title-main">CHEK2</span> Protein-coding gene in humans

CHEK2 is a tumor suppressor gene that encodes the protein CHK2, a serine-threonine kinase. CHK2 is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Mutations to the CHEK2 gene have been linked to a wide range of cancers.

<span class="mw-page-title-main">Cyclin-dependent kinase 1</span> Mammalian protein found in Homo sapiens

Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine protein kinase, and is a key player in cell cycle regulation. It has been highly studied in the budding yeast S. cerevisiae, and the fission yeast S. pombe, where it is encoded by genes cdc28 and cdc2, respectively. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.

<span class="mw-page-title-main">CHEK1</span> Protein-coding gene in humans

Checkpoint kinase 1, commonly referred to as Chk1, is a serine/threonine-specific protein kinase that, in humans, is encoded by the CHEK1 gene. Chk1 coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chk1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle.

<span class="mw-page-title-main">Cyclin B1</span> Protein-coding gene in the species Homo sapiens

G2/mitotic-specific cyclin-B1 is a protein that in humans is encoded by the CCNB1 gene.

<span class="mw-page-title-main">CDC25A</span> Protein-coding gene in the species Homo sapiens

M-phase inducer phosphatase 1 also known as dual specificity phosphatase Cdc25A is a protein that in humans is encoded by the cell division cycle 25 homolog A (CDC25A) gene.

<span class="mw-page-title-main">CDC25B</span> Protein-coding gene in the species Homo sapiens

M-phase inducer phosphatase 2 is an enzyme that in humans is encoded by the CDC25B gene.

<span class="mw-page-title-main">Wee1-like protein kinase</span> Protein-coding gene in the species Homo sapiens

WEE1 homolog , also known as WEE1, is a protein which in humans is encoded by the WEE1 gene.

<span class="mw-page-title-main">CDC25C</span> Protein-coding gene in the species Homo sapiens

M-phase inducer phosphatase 3 is an enzyme that in humans is encoded by the CDC25C gene.

<span class="mw-page-title-main">MDC1</span> Protein-coding gene in the species Homo sapiens

Mediator of DNA damage checkpoint protein 1 is a 2080 amino acid long protein that in humans is encoded by the MDC1 gene located on the short arm (p) of chromosome 6. MDC1 protein is a regulator of the Intra-S phase and the G2/M cell cycle checkpoints and recruits repair proteins to the site of DNA damage. It is involved in determining cell survival fate in association with tumor suppressor protein p53. This protein also goes by the name Nuclear Factor with BRCT Domain 1 (NFBD1).

<span class="mw-page-title-main">PKMYT1</span> Protein-coding gene in the species Homo sapiens

Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase also known as Myt1 kinase is an enzyme that in humans is encoded by the PKMYT1 gene.

<span class="mw-page-title-main">Wee1</span> Nuclear protein

Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe. Wee1 has a molecular mass of 96 kDa and is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1. Wee1 has homologues in many other organisms, including mammals.

<span class="mw-page-title-main">Meiotic recombination checkpoint</span>

The meiotic recombination checkpoint monitors meiotic recombination during meiosis, and blocks the entry into metaphase I if recombination is not efficiently processed.

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

References

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