The cell cycle has three major checkpoints: one in G1 (the gap before DNA replication), one in G2 (the gap after replication but before division), and one during M phase (mitosis itself). There is also a lesser-known checkpoint that operates during S phase, when DNA is actively being copied. Each checkpoint evaluates different conditions and blocks the cell from moving forward until those conditions are met.
The G1 Checkpoint (Restriction Point)
The first major checkpoint sits in the G1 phase, near the boundary where the cell would commit to copying its DNA. This is often called the restriction point, and it’s the most consequential decision the cell makes: move forward toward division, or pull out of the cycle entirely into a resting state called G0. Most cells in your body are sitting quietly in G0 right now. Quiescent cells, like resting immune cells, are held there by proteins that suppress the genes needed for cell cycle entry. To re-enter the cycle, they need sustained stimulation, sometimes for several hours, from growth signals.
At the G1 checkpoint, the cell evaluates three broad categories of information:
- Growth factor signals. External chemical signals tell the cell whether the body actually needs more cells. Without these growth factors, the cell exits into G0 rather than proceeding. In cancer, mutations can mimic these signals, pushing cells past the restriction point without permission.
- Nutrient and energy status. A nutrient-sensing system in late G1 checks whether the cell has enough raw materials and energy to double in size before committing to divide.
- DNA integrity. If the cell’s DNA is damaged, the protein p53 accumulates and activates a braking system. p53 triggers production of proteins that block the molecular engines driving the cell forward. This gives the cell time to repair damage or, if the damage is too severe, to self-destruct through programmed cell death.
The molecular engine at this checkpoint runs on partnerships between proteins called cyclins and their partner enzymes. Cyclin D pairs with CDK4 and CDK6, and together they begin disabling the tumor suppressor protein Rb. Rb normally acts like a lock on cell cycle genes. As cyclin D/CDK4/6 weakens Rb’s grip, another pair, cyclin E with CDK2, finishes the job. Once Rb is fully disabled, the cell crosses the restriction point and no longer needs external growth signals to finish the current cycle.
The Intra-S Phase Checkpoint
While DNA is being replicated during S phase, a dedicated surveillance system monitors the copying process in real time. This intra-S checkpoint doesn’t get as much attention as the big three, but it plays a critical role in catching problems as they happen. The replication machinery physically encounters every base in the genome, making it an extraordinarily sensitive detector of damage. Studies have shown that an extremely small amount of a DNA-damaging chemical, as little as 0.005% of one common laboratory mutagen, is enough to trigger this checkpoint during S phase, while the same amount causes no response outside of replication.
When damage is detected at a replication fork (the point where DNA is being unwound and copied), the checkpoint responds in three ways: it slows down the progression of active replication forks, it prevents new replication origins from firing up, and it adjusts gene activity. Of these, regulating fork progression appears to be the most important function. Notably, a single checkpoint pathway handles the response to many different types of DNA damage during S phase, rather than having separate systems for each kind of lesion.
The G2 Checkpoint
After DNA replication is complete, the cell enters G2 and faces another inspection before it can begin dividing. The G2 checkpoint verifies that the entire genome was copied correctly and that no DNA damage remains unrepaired. This checkpoint is ancient, conserved from single-celled yeast all the way to humans.
The central question at G2 is simple: should the cell activate the molecular machinery that drives it into mitosis? That machinery centers on CDK1 paired with cyclin A and later cyclin B. If DNA damage exists, the cell keeps CDK1 locked in an inactive state. Here’s how it works: damaged DNA generates stretches of exposed single-stranded DNA, either directly from the lesion or from enzymes chewing back around a break. A protein called RPA rapidly coats these exposed stretches, creating a landing platform for sensor proteins. These sensors activate a signaling cascade that ultimately switches on a kinase called Chk1, which keeps the mitotic engine switched off.
The cell also checks its size at G2, just as it did in G1. If the cell hasn’t grown enough, it waits. p53 contributes here too. When activated by damage, p53 triggers several parallel braking pathways that prevent the mitotic cyclin B/CDK1 complex from forming or functioning. One pathway blocks CDK1 activity directly, another disrupts the physical pairing of cyclin B with CDK1, and a third sequesters an activating enzyme so it can’t do its job. p53 even represses the genes that produce cyclin B and CDK1 in the first place.
The Spindle Assembly Checkpoint (M Phase)
The final major checkpoint operates during mitosis itself, specifically at the transition from metaphase to anaphase. By this point, chromosomes have condensed and the spindle, a scaffold of protein fibers, has formed. The spindle assembly checkpoint ensures that every single chromosome is properly attached to spindle fibers from opposite poles of the cell before the chromosomes are pulled apart.
The mechanism is elegant. Any kinetochore (the attachment point on a chromosome) that lacks a stable connection to spindle fibers, or that is attached but not under mechanical tension, generates a “wait” signal. Even one unattached kinetochore is enough to halt the entire process. The checkpoint proteins accumulate at these unattached sites and block the activation of a large molecular machine called the Anaphase-Promoting Complex (APC/C). The APC/C is an enzyme that tags specific proteins for destruction. Its two key targets are securin, which holds sister chromosomes together, and cyclin B, which keeps the cell in mitosis.
Once every chromosome achieves proper bipolar attachment and the kinetochores come under tension from being pulled toward opposite poles, the checkpoint signal shuts off. The APC/C activates, tags securin and cyclin B for destruction, and the cell splits its chromosomes and proceeds to divide.
What Happens When Checkpoints Fail
Checkpoint failure is one of the defining features of cancer. Proteins involved in the G1/S checkpoint are inactivated in the majority of human cancers. The most prominent example is p53, which is disabled in roughly 50% of all human cancers. Without p53, cells with damaged DNA sail through G1 and G2 without stopping, accumulating mutations with each division.
Failures at the G2 checkpoint are also common in cancer, and these defects may explain why some tumors resist radiation and chemotherapy. Both treatments work by damaging DNA, so a cell that can’t detect that damage and stop dividing will keep proliferating despite treatment.
Spindle checkpoint failures lead to a different kind of problem: cells that divide with the wrong number of chromosomes, a condition called aneuploidy. Colorectal cancers, for example, experience chromosome segregation errors roughly once every 100 cell divisions, leading to widespread chromosomal instability. Some of these cancers carry mutations in spindle checkpoint proteins that cause cells to exit mitosis prematurely, before chromosomes are properly sorted. Whether this chromosomal instability directly causes cancer or is a byproduct of it remains an open question, but the link between checkpoint failure and tumor development is well established.