G2/M Checkpoint: DNA Damage Regulation in the Cell Cycle
The G2/M checkpoint is a crucial control mechanism that ensures a cell is ready to enter mitosis and divide. This checkpoint monitors for DNA damage and incomplete DNA replication, halting cell cycle progression if the genome is not fully intact and providing time for repair and replication completion. It is essential for preserving genomic stability and safeguarding against mutations that can lead to cancer.
Pathway Summary
G2/M checkpoint is the second checkpoint within the cell cycle. This checkpoint prevents cells with damaged DNA from entering the M phase, while also pausing so that DNA repair can occur. This regulation is important to maintain genomic stability and prevent cells from undergoing malignant transformation.Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and rad3 related (ATR) are key kinases that respond to DNA damage. ATR responds to UV damage, while ATM responds to DNA double-strand breaks (DSB). ATM and ATR activate kinases Chk1 and Chk2 which in turn inhibit Cdc25, the phosphatase that normally activates Cdc2. Cdc2, a cyclin-dependent kinase, is a key molecule that is required for entry into M phase. It requires binding to cyclin B1 for its activity.The tumor suppressor gene p53 is an important molecule in G2/M checkpoint regulation. ATM, ATR and Chk2 contribute to the activation of p53. Further, p19Arf functions mechanistically to prevent MDM2's neutralization of p53. Mdm4 is a transcriptional inhibitor of p53. DNA damage-induced phosphorylation of Mdm4 activates p53 by targeting Mdm4 for degradation. Well known p53 target genes like Gadd45 and p21 are involved in inhibiting Cdc2. Another p53 target gene, 14-3-3σ, binds to the Cdc2-cyclin B complex rendering it inactive. Repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. In this way, numerous checks are enforced before a cell is allowed to enter the M phase.
Cell Cycle: G2/M DNA Damage Checkpoint Regulation Genes list
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Overview of G2/M checkpoint regulation
G2 phase is the final stage of the cell cycle before the cell enters mitosis (M phase). During G2 (Gap 2), the cell continues to grow and produce proteins necessary for chromosome segregation and cell division. It also assesses the integrity of the DNA replicated during S phase, ensuring the genome is complete and undamaged.
The G2/M checkpoint acts as a critical control point at the end of this phase, pausing the cycle if errors or damage are detected. Its primary role is to prevent cells from entering mitosis with broken chromosomes or unreplicated DNA, thereby maintaining genomic stability.
The G2/M checkpoint monitors several key conditions to determine if a cell is ready for mitosis. At a basic level, the checkpoint ensures proper cell size and energy reserves, both of which are necessary for the energetically demanding process of mitosis.
How does the G2 checkpoint ensure DNA integrity?
The checkpoint evaluates whether the cell’s DNA is competent to generate two faithfully replicated daughter cells. It does this in two ways. First, the checkpoint verifies that DNA replication is fully completed – checking for stalled replication forks or under-replicated regions that signal replication is incomplete. Secondly, it checks for DNA damage. If DNA damage is not detected and all other signals concur that replication is complete and the cell has the needed resources, the cell will be allowed to move into M phase. If DNA damage is detected, the cell cycle process will be paused, allowing for repairs to be completed before resuming. If the DNA damage is too severe, mitotic catastrophe may occur.
G2/M checkpoint regulation in response to DNA damage
The G2/M checkpoint in cell cycle serves as a critical surveillance mechanism that prevents cells from entering mitosis when DNA damage is present or replication is incomplete. The G2/M checkpoint actively monitors the integrity of the cell’s DNA, initiating a signaling cascade that pauses cell cycle progression when it is compromised. The process, which shares many components with the G1/S checkpoint earlier in the cell cycle, is generally broken into four steps: sensing DNA damage, activating checkpoint kinases, cell cycle arrest and finally, resolution and resumption of the cell cycle.
DNA damage sensing: ATR and ATM activation
As with the G1/S checkpoint response earlier in the cell cycle, activation of ATR (ATM and Rad3-related) and of ATM (ataxia-telangiectasia mutated) phosphatidylinositol 3-kinase–like kinases (PIKKs) initiates the DNA damage response. ATR responds mainly to single stranded DNA (ssDNA) lesions and is recruited with help from the ATRIP protein and RPA-coated ssDNA, while ATM is primarily activated by double-strand breaks and is recruited to the damaged site via the MRN complex.
In the G2/M context, ATR also responds to replication stress by detecting persistent single-stranded DNA at stalled replication forks, preventing premature chromosome condensation. ATM, in addition to activating checkpoint signaling, helps recruit and coordinate repair complexes at double-strand break sites, linking checkpoint control directly to DNA repair machinery.
Checkpoint kinase activation and CDC25C inhibition
Once again, similar to the earlier G1/S checkpoint response, activated ATM and ATR phosphorylate and activate checkpoint kinases, particularly CHK1 (downstream of ATR) and CHK2 (downstream of ATM). At the G2/M checkpoint, these kinases prevent mitotic entry by phosphorylating CDC25C, promoting its binding to 14-3-3σ, which sequesters CDC25C in the cytoplasm.
Additional regulation occurs through the inhibition of other Cdc25 phosphatase family members, such as Cdc25A, and through phosphorylation of Wee1 kinase, which maintains CDK1 in an inactive phosphorylated state. This dual control ensures that the Cyclin B/CDK1 complex remains inactive until all checkpoint conditions are cleared.
Cell cycle arrest: CDC25C and Cyclin B/CDK1 inhibition
Under normal conditions, CDC25C translocates to the nucleus, where it removes inhibitory phosphates from the CDK1/Cyclin B complex, the master regulator of mitotic entry. As CDK1/Cyclin B becomes activated, progression into mitosis is allowed. When CDC25C is sequestered in the cytoplasm, it is prevented from dephosphorylating CDK1/Cyclin B, keeping it in an inactive state and pausing cell cycle progression to allow time for DNA repair and/or completion of replication.
This arrest is reinforced by the continued activity of Wee1 kinase and the suppression of pro-mitotic transcription factors such as FOXM1, ensuring that no preparatory mitotic events proceed prematurely.
Resolution and progression to mitosis
Once DNA damage is repaired and replication is complete, ATM/ATR signaling diminishes, leading to degradation of CHK1/CHK2 and release of CDC25C from 14-3-3σ. CDC25C can then enter the nucleus, remove inhibitory phosphates from CDK1 and trigger full activation of CDK1/Cyclin B, allowing the cell to enter mitosis.
Importantly, resolution of the checkpoint also requires restoration of proper spindle assembly signals via the spindle assembly checkpoint, ensuring that chromosomal segregation will proceed accurately in M phase.
This tightly regulated pathway acts as a safeguard, preventing premature mitotic entry. Disruption in any part of this pathway, particularly overactive CDC25C or loss of 14-3-3σ, can result in defective mitosis leading to chromosome mis-segregation and tumorigenesis.
Consequences of defective G2/M checkpoint regulation
Defective regulation of the G2/M checkpoint undermines the cell’s ability to prevent mitotic entry in the presence of DNA damage or incomplete replication, allowing cells to divide with damaged or unstable genomes. This results in the accumulation of mutations and chromosomal aberrations, driving genomic instability, which is a key contributor to tumorigenesis. Additionally, the inability to properly enforce this checkpoint removes a crucial barrier to uncontrolled cell proliferation, further promoting cancer progression.
In some cases, premature entry into mitosis with unrepaired damage can lead to mitotic catastrophe, a form of cell death characterized by aberrant mitosis and chromosome mis-segregation. While mitotic catastrophe can serve as a tumor-suppressive mechanism, it may instead contribute to aneuploidy and therapeutic resistance if avoided or tolerated by cancer cells.
Importantly, many cancer treatments rely on functional G2/M checkpoints to halt cell division in response to DNA-damaging agents. Cells with defective checkpoint control often fail to arrest, evade apoptosis and develop resistance to therapy, limiting treatment effectiveness and contributing to disease relapse. This has driven interest in developing targeted inhibitors of checkpoint kinases (for example, CHK1 inhibitors) as a means of selectively sensitizing cancer cells with pre-existing checkpoint defects to DNA-damaging therapies.
References
- Liu K, Zheng M, Lu R, Du J, Zhao Q, et al. The role of CDC25C in cell cycle regulation and clinical cancer therapy: A systematic review. Cancer Cell Int. 2020;20:213.
- Lang F, Cornwell JA, Kaur K, Elmogazy O, Zhang W, et al. Abrogation of the G2/M checkpoint as a chemosensitization approach for alkylating agents. Neuro Oncol. 2024;26(6):1083–1096.