Chromosomes are duplicated during S phase, the “synthesis” stage of the cell cycle. S phase sits between two gap phases (G1 and G2) and is followed by M phase, when the cell physically divides. But calling S phase “the copying step” undersells what actually happens: the cell doesn’t just photocopy its DNA. It rebuilds the protein packaging around that DNA, copies chemical tags that control gene activity, and even duplicates structures needed for cell division, all while running quality-control checks that can slam the brakes if something goes wrong.
The Four Stages in Order
The cell cycle proceeds through a fixed sequence of phases that every dividing cell follows. These stages, always occurring in the same order, are G1, S, G2, and M. G1 is a growth phase where the cell increases in size and prepares for DNA synthesis. S phase is when the genome gets copied. G2 is a second gap phase where the cell checks its work and continues growing. M phase (mitosis) is the dramatic finale, where the duplicated chromosomes are pulled apart and the cell splits in two.
1PubMed Central. A Journey through Time on the Discovery of Cell Cycle RegulationThe whole cycle can take anywhere from about eight hours in a fast-dividing embryonic cell to well over 24 hours in a typical adult human cell. S phase itself usually accounts for roughly six to eight hours of that total. The time isn’t trivial: a human cell has to copy about six billion base pairs of DNA, and it has to do so with very few errors.
Getting Ready to Copy in G1
Before S phase even begins, the cell does critical prep work during G1. This process, called origin licensing, involves loading protein complexes onto specific sites along each chromosome. These sites will become the starting points for DNA replication. The protein complexes loaded there, known as pre-replicative complexes, essentially give the cell permission to duplicate that stretch of DNA during S phase.
2Nature Structural & Molecular Biology. Mechanisms for licensing origins of DNA replication in eukaryotic cellsOrigin licensing is tightly restricted to G1. Once a cell enters S phase and starts copying its DNA, it cannot load new pre-replicative complexes. This “once and only once” rule is one of the most important safety features in cell biology, because it prevents the same stretch of chromosome from being copied twice. If a region were duplicated more than once in a single cycle, the daughter cells would end up with extra copies of genes, which can be catastrophic.
3PubMed Central. Efficiency and equity in origin licensing to ensure complete DNA replicationThousands of origins get licensed across the genome, but not all of them will actually fire during S phase. Many serve as backups. If a replication fork stalls or fails, a nearby dormant origin can activate to finish the job. The cell essentially over-prepares in G1 so that S phase has a safety net.
What Happens During S Phase
When S phase begins, the licensed origins start firing. Each active origin launches two replication forks that move in opposite directions along the chromosome, unzipping the double helix and using each strand as a template to build a new complementary strand. Replication origins don’t all fire simultaneously. Some activate early in S phase, others in the middle, and some don’t fire until late S phase. This staggered schedule means the cell doesn’t need to supply all its replication machinery at once.
4PubMed Central. Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cellsAs the two forks from each origin move outward, they eventually meet forks traveling from neighboring origins. When they collide, the replication machinery disassembles and the newly synthesized stretches are joined together. By the end of S phase, the result is two complete copies of each chromosome, called sister chromatids, held together by a ring-shaped protein complex. This holding-together step happens right alongside DNA copying: as the replication fork passes through a region, the cohesin complex clamps the two new sister chromatids to each other.
5PubMed Central. A replication fork determinant for the establishment of sister chromatid cohesionThat cohesion is essential. If the sister chromatids drifted apart immediately, the cell would have no reliable way to organize them for proper segregation during mitosis. The cohesin rings keep them paired until the moment they need to separate.
Not Everything Replicates at the Same Time
The timing of when different chromosomal regions replicate during S phase is surprisingly ordered, and it tracks with how those regions are used. Actively transcribed regions with a more open, accessible structure tend to replicate early in S phase. Tightly packed, gene-poor regions tend to replicate late. Research in human and mouse cells has confirmed that earlier-replicated regions show greater physical accessibility at the level of their protein packaging, and later-replicated regions are more compact and less mobile.
6PubMed Central. Replication-dependent histone labeling dissects the physical properties of euchromatin/heterochromatin in living human cellsThe general rule holds across most organisms, but there are interesting exceptions. In fission yeast, for instance, the highly condensed regions around centromeres replicate early rather than late, even though they have the structural hallmarks of tightly packed chromatin. Only the chromosome tips replicate late in that organism.
7PubMed Central. Early-replicating heterochromatinWhy should the timing matter at all? One reason is practical: early-replicating regions sit in parts of the nucleus that are more accessible to the replication machinery. Another reason is that replication timing is closely linked to which genes are turned on or off in a given cell type. The replication-timing program differs between, say, a liver cell and a brain cell, reflecting their distinct gene-activity profiles. When the timing program goes haywire, it can contribute to disease.
Duplicating More Than Just DNA
If S phase only copied the sequence of bases in DNA, it would miss half the story. DNA in your cells isn’t bare. It’s wrapped around protein spools called histones, and those histones carry chemical modifications that help determine which genes are active. During S phase, the cell has to rebuild this packaging on both copies of each chromosome. Newly replicated DNA gets loaded with a mix of old histones recycled from the parent strand and fresh histones manufactured for the occasion.
8Trends in Biochemical Sciences. Replication-Coupled Nucleosome Assembly in the Passage of Epigenetic Information and Cell IdentityThis assembly process is tightly coupled to the speed of DNA copying. If the cell doesn’t produce enough histones, DNA replication actually slows down to wait.
9PubMed Central. Histone supply regulates S phase timing and cell cycle progressionOn top of histone packaging, the cell also copies DNA methylation marks during S phase. DNA methylation is a chemical tag added directly to certain bases in the DNA sequence, and it plays a major role in silencing genes. A specialized complex is recruited onto newly replicated DNA during S phase to copy the methylation pattern from the old strand to the new one, preserving the cell’s gene-silencing instructions for both daughter cells.
10PubMed Central. Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycleThis is why S phase isn’t just about genetics in the narrow sense. It’s also about epigenetics, the layer of information above the DNA sequence that tells a liver cell to stay a liver cell and a neuron to stay a neuron. Errors in copying this epigenetic information during S phase can change a cell’s identity, and that can contribute to cancer and other diseases.
Centrosomes Duplicate in Parallel
While the chromosomes are being copied, something else is getting duplicated at the same time: the centrosome, a small structure that will later organize the fibers that pull chromosomes apart during mitosis. Each cell enters S phase with one centrosome and needs two by the time it reaches mitosis. The duplication of centrosomes is coordinated with DNA replication through shared molecular signals.
11PubMed. Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extractsThe coupling between centrosome duplication and DNA replication is enforced by the same enzyme activity that triggers the start of S phase. Some of the proteins involved in licensing DNA replication origins also appear to regulate centrosome copying, which makes biological sense: you want exactly two centrosomes ready at the same time that you have two complete sets of chromosomes.
12PubMed. Cyclin E-dependent localization of MCM5 regulates centrosome duplicationWhen this coordination breaks down, cells can end up with too many centrosomes, which leads to chaotic chromosome segregation during mitosis. Extra centrosomes are a common feature of cancer cells and are thought to drive the genomic instability that makes tumors progressively more dangerous over time.
13Leukemia. Centrosome replication, genomic instability and cancerQuality Control During and After Copying
Cells don’t blindly barrel through S phase hoping for the best. Multiple checkpoint systems monitor the replication process and intervene when things go wrong. If a replication fork stalls, perhaps because it runs into damaged DNA or a tightly bound protein, the cell activates what’s called the intra-S phase checkpoint. This system directly targets the enzyme that unwinds DNA ahead of the replication fork, reducing its activity and slowing down replication across the genome until the problem is resolved.
14PubMed Central. The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomesSlowing down replication might sound counterproductive, but it prevents a bad situation from getting worse. A stalled fork that isn’t stabilized can collapse, creating double-strand breaks in the DNA, which are one of the most dangerous forms of DNA damage. The checkpoint buys time for repair machinery to fix the problem before the fork falls apart.
After S phase ends and the cell enters G2, additional verification happens. One critical task is making sure the two sister chromatids can physically separate. An enzyme that untangles intertwined DNA strands is essential for this separation, and when that enzyme is blocked or missing, cells activate a G2 checkpoint that prevents entry into mitosis. If the cell somehow reaches mitosis with tangled chromatids anyway, a further checkpoint at the end of cell division can block the final splitting of the cell to prevent the damage from propagating.
15PubMed Central. Cell cycle responses to Topoisomerase II inhibitionWhen Duplication Goes Wrong
Cancer is, in many ways, a disease of mismanaged chromosome duplication. When genes that drive cell growth (oncogenes) become overactive, they can push cells into S phase before the cell is properly prepared, creating what researchers call replication stress. This stress manifests in several ways: origins fire at the wrong times, replication forks collide with the gene-reading machinery, the building blocks for new DNA run short, and damaging chemical byproducts accumulate. The result is an error-prone copying process that introduces mutations and structural rearrangements into the genome.
16PubMed Central. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into PlaceReplication stress is considered one of the earliest drivers of genomic instability in cancer development. It can occur before a cell even looks abnormal under a microscope, and it feeds a vicious cycle: replication errors create mutations, some of which disable the very checkpoint systems meant to catch replication errors, leading to even more errors in subsequent cell divisions.
Drugs That Exploit S Phase
Because cancer cells divide more frequently than most normal cells, they spend more time in S phase, making this stage an attractive target for chemotherapy. One of the oldest and most widely used examples is hydroxyurea, which blocks the enzyme that converts the building blocks of RNA into the building blocks of DNA. Without those DNA building blocks, S phase grinds to a halt.
17PubMed Central. Hydroxyurea-The Good, the Bad and the UglyBeyond cancer treatment, hydroxyurea has found a major role in managing sickle cell disease, where it works through a different mechanism (boosting production of a fetal form of hemoglobin). Its ability to arrest cells in S phase also makes it an indispensable tool in laboratory research for synchronizing cell populations so that scientists can study specific phases of the cell cycle in isolation.
Other S-phase-specific drugs, like gemcitabine and cytarabine, mimic the building blocks of DNA. They get incorporated into the growing DNA strand during replication and then disrupt the process, either stalling the replication fork or introducing lethal errors. These drugs work specifically against cells actively copying their DNA, which means they hit rapidly dividing cancer cells harder than most resting cells.
18PubMed. Hydroxyurea: a key player in cancer chemotherapyResearch has even explored flipping this specificity on its head: instead of killing cells in S phase, you can protect normal cells from S-phase-targeting drugs. In laboratory experiments, activating a tumor-suppressor protein called p53 before giving gemcitabine caused normal cells to arrest in G1, keeping them out of S phase and away from the drug’s toxic effects. Cancer cells that had already lost functional p53 kept entering S phase and remained vulnerable.
19PubMed. Nongenotoxic p53 activation protects cells against S-phase-specific chemotherapyHow Scientists Tell Which Phase a Cell Is In
Figuring out whether a cell is in G1, S, G2, or M is a routine part of cell biology research. The classic approach uses flow cytometry, a technique that measures the amount of DNA in individual cells as they stream past a detector. Cells in G1 have the standard amount of DNA. Cells that have finished S phase and are in G2 or M have exactly double that amount. Cells caught mid-S phase fall somewhere in between, because they’ve copied some of their genome but not all of it.
20PubMed Central. Assaying cell cycle status using flow cytometryFor a more direct look at S phase specifically, researchers can feed cells a modified DNA building block that gets incorporated into newly synthesized DNA. A widely used version of this approach relies on a molecule called EdU, which cells pick up and insert into their DNA during replication. After fixation, a chemical reaction tags every EdU molecule with a fluorescent marker, lighting up cells that were actively copying their DNA at the time of the experiment.
21PubMed Central. A chemical method for fast and sensitive detection of DNA synthesis in vivoCombining EdU labeling with measurements of other cell-cycle proteins lets researchers map not just whether a cell is in S phase, but how far through S phase it has progressed and which regulatory proteins are present at each point.
22PubMed Central. Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometryCells That Copy Without Dividing
Not every cell that duplicates its chromosomes goes on to divide. Some cells run through S phase, double their genome, and then skip mitosis entirely, ending up with twice the normal chromosome content. If this happens repeatedly, a cell can accumulate many copies of its genome, becoming what biologists call polyploid. This process, known as endoreplication, isn’t an accident. It’s a deliberate developmental strategy used by many organisms.
23PubMed Central. Endoreplication: The Good, the Bad, and the UglyIn plants, endoreplication is extremely common and helps cells grow large without the metabolic cost of building new cell membranes. The fleshy part of a tomato fruit, for example, contains cells with dozens of genome copies. In mammals, certain liver cells and the giant cells that produce blood platelets use endoreplication for similar reasons: bigger cells with more copies of the genome can produce proteins at a higher rate. Even some insect tissues rely heavily on endoreplication during development.
Bacteria Play by Different Rules
The G1-S-G2-M cycle described above applies to eukaryotic cells. Bacteria, which lack a nucleus and have a single circular chromosome, handle duplication differently. Fast-growing bacteria face a particular challenge: their chromosome takes longer to copy than the time between cell divisions. The solution is to start a new round of replication before the previous one has finished, resulting in cells that carry partially duplicated chromosomes at all times.
24PubMed Central. Multifork chromosome replication in slow-growing bacteriaThis overlapping replication, called multifork replication, means that genes near the origin of replication are present in more copies than genes near the terminus. Bacteria actually exploit this asymmetry: genes that need to be expressed at high levels, like those encoding the protein-building machinery, tend to sit near the origin where they exist in extra copies during rapid growth. It’s an elegant bit of evolutionary engineering, and a reminder that the question “when are chromosomes duplicated?” has a fundamentally different answer in organisms that lack a defined cell cycle.