S-phase is the period when a cell copies its entire genome so that each daughter cell will inherit a complete set of DNA after division. But DNA duplication is only the headline act. During the same window, the cell also rebuilds the protein packaging around its new DNA, copies chemical tags that control which genes stay on or off, glues freshly made chromosome copies together, duplicates key structural components, extends the protective caps on chromosome ends, and runs a real-time surveillance system to catch errors before they become permanent. All of this happens in a coordinated rush that, in a typical human cell, lasts roughly six to eight hours.
Firing Up Thousands of Replication Origins
A human cell does not start copying its DNA from a single spot and work its way to the end. Instead, the genome contains tens of thousands of licensed origins, specific sites where copying can begin. During S-phase, clusters of these origins fire in a regulated sequence. Early-firing origins tend to sit in regions of the genome that are more open and actively transcribed, while late-firing origins are usually found in more tightly packed, less active regions. In fission yeast, researchers found a notable exception: certain condensed regions around centromeres and silent mating-type genes actually replicate early, while the condensed regions at chromosome tips still replicate late.1Genes & Development. Early-replicating heterochromatin So the rule that open DNA replicates first and closed DNA replicates last holds broadly, but evolution has carved out important exceptions where early copying serves a structural purpose.
The timing of origin firing is not random. A key regulated step involves the assembly of what is called a pre-initiation complex, a cluster of proteins that commits an origin to fire. Multiple pathways converge on a kinase called DDK, which acts as a trigger. Meanwhile, a protein called Rif1 acts as a brake on late-firing origins, keeping them dormant until the right moment. Origins also compete with each other for a limited pool of firing factors, which helps enforce the staggered schedule.2Europe PMC. Origin Firing Regulations to Control Genome Replication Timing This layered control ensures that the cell does not try to copy everything at once, which would overwhelm its supply of raw materials and error-checking machinery.
The Enzymes That Build New DNA
Once an origin fires, the double helix unwinds and two replication forks move outward in opposite directions. At each fork, the two strands of the original DNA are copied by different enzymes. One strand, called the leading strand, is copied continuously in the direction the fork moves. The other, the lagging strand, is copied in short fragments that are later stitched together. These two jobs are handled by distinct polymerases. Work in frog egg extracts showed that the polymerase called Pol delta is essential for lagging-strand synthesis and that Pol epsilon cannot substitute for it in that role. Depleting either enzyme slowed DNA copying and caused short, unfinished fragments to pile up, but losing Pol delta produced a more severe defect, with persistent single-stranded gaps appearing on the lagging strand.3PubMed. Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts
A third polymerase, Pol alpha, lays down a short RNA-DNA primer at each origin and at the start of each lagging-strand fragment. This primer is later removed and replaced with properly synthesized DNA. Together, these three polymerases form an assembly line, but they also cross-check each other. Genetic experiments in yeast found that Pol epsilon can proofread certain errors made by Pol alpha and Pol delta during replication, adding an extra layer of quality control.4PubMed Central. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations The result is a remarkably accurate copying process, with roughly one uncorrected error per billion nucleotides in a well-functioning cell.
Rebuilding Chromatin on Freshly Copied DNA
DNA in a cell is not naked. It wraps around spool-like protein complexes called nucleosomes, and this packaging profoundly affects which genes are accessible. When the replication fork barrels through, it strips nucleosomes off the parental DNA. The cell has to reassemble them on both daughter strands almost immediately, or risk losing the gene-activity patterns encoded in the chromatin structure.
To meet this demand, the cell dramatically ramps up histone production during S-phase. Eukaryotic cells use specialized machinery to translate a particular class of histone messenger RNAs that are tightly coupled to DNA replication, ensuring that the supply of histone proteins surges exactly when it is needed.5Cell Death Discovery. ZCCHC4 promotes replication-dependent histone mRNA translation through interaction with the eIF3 complex A dedicated assembly factor called CAF-1 then deposits fresh histones onto newly synthesized DNA right behind the moving fork. This process requires CAF-1 to bind DNA directly; without that binding, efficient assembly of new nucleosomes during replication stalls.6PubMed Central. Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1 Old histones displaced by the fork are also recycled onto the daughter strands, carrying some of the chemical modifications from the parent cell and helping preserve the cell’s identity.
Copying Epigenetic Marks
Beyond the sequence of DNA letters, cells carry a second layer of information in the form of chemical tags, most commonly methyl groups attached to cytosine bases. These tags help silence genes that should stay off in a given cell type. When the replication fork passes through, only the old strand retains its methyl groups. The new strand is bare, creating a temporary half-methylated state.
The cell corrects this within minutes. A protein called UHRF1 recognizes and binds these half-methylated sites through a specialized domain, then recruits the maintenance methyltransferase DNMT1 to the replication fork.7Nucleic Acids Research. The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity UHRF1 also tags nearby histones with ubiquitin marks that help anchor DNMT1 at the right spots. This entire interaction is S-phase-dependent: UHRF1 binds DNMT1 during S-phase but not at other times, acting as a cell-cycle switch that confines methylation maintenance to the window when it is needed.8PubMed Central. S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance Both DNMT1 and UHRF1 physically concentrate at active replication sites during S-phase, ensuring that the methylation pattern is faithfully restored on each daughter strand.9Journal of Biological Chemistry. The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1
If this copying process fails, a cell can gradually lose its methylation pattern over successive divisions. That kind of drift is a hallmark of aging cells and a common feature of cancer, where silenced tumor-suppressor genes can be accidentally reactivated or active genes silenced in the wrong context.
Gluing Sister Chromatids Together
As each stretch of DNA is copied, the two resulting sister chromatids need to be held together until the cell is ready to pull them apart during mitosis. This physical link, called cohesion, is established during S-phase and not before or after. Ring-shaped protein complexes called cohesins encircle the sister chromatids, but they need to be locked in place by an enzyme called ESCO2, which acetylates a specific cohesin subunit. This acetylation is what commits the cohesin ring to hold the two sisters together rather than simply sliding off.10PubMed Central. Symmetric control of sister chromatid cohesion establishment
The timing is precise: accessory factors facilitate the arrival of ESCO2 on chromatin during S-phase, which then triggers the acetylation of the cohesin subunit SMC3.11Nucleic Acids Research. Symmetric control of sister chromatid cohesion establishment ESCO2 belongs to a conserved protein family found across many species, and mutations in the human gene cause Roberts syndrome, a developmental disorder characterized by limb malformations and growth problems. The connection makes sense: if cohesion is not properly established during S-phase, chromosomes can mis-segregate during division, leading to cells with the wrong number of chromosomes.12PubMed. Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion
Centriole Duplication and Telomere Extension
S-phase is also the window for duplicating centrioles, small cylindrical structures that organize the cell’s internal skeleton and later help form the spindle poles needed to pull chromosomes apart. Each cell enters S-phase with two centrioles, and during this period each one sprouts a new daughter centriole at its base. Overexpression of the kinase Plk4 in human cells showed that new centriole assembly occurs specifically in S-phase and the following G2 phase; cells in G1 did not form the characteristic rosette-like assembly intermediates.13Developmental Cell. Plk4-Induced Centriole Biogenesis in Human Cells Restricting duplication to S-phase prevents a cell from accumulating extra centrioles, which can cause abnormal cell divisions.
Meanwhile, chromosome ends require their own maintenance. Telomeres, the repetitive DNA caps that protect chromosome tips from degrading, shorten slightly with each round of replication because the copying machinery cannot fully replicate the very end of a linear chromosome. The enzyme telomerase counteracts this by adding new repeats. In human cells, telomerase is recruited to subsets of telomeres specifically during S-phase, with its presence peaking at mid-S-phase.14PubMed Central. Cell cycle-regulated trafficking of human telomerase to telomeres This coordination ensures that the enzyme extends telomeres while the replication fork is still nearby, rather than at some arbitrary later time.
When the Fork Meets a Transcription Machine
DNA replication and gene transcription both move along the same DNA template, which means collisions are inevitable. When a replication fork runs into an RNA polymerase that is actively transcribing a gene, the result is a conflict that can stall the fork and threaten the integrity of the genome. Cells rely on multiple mechanisms to prevent, tolerate, and resolve these encounters.15PubMed Central. Conflict Resolution in the Genome: How Transcription and Replication Make It Work
One recently discovered mechanism involves a protein complex called Integrator, which normally helps process RNA transcripts. Researchers found that Integrator physically interacts with the replication machinery at active forks and promotes the removal of RNA polymerase from the fork’s path. Without Integrator, forks slow down and cells accumulate chromosome breaks and other hallmarks of genome instability.16PubMed Central. Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability This finding illustrates that S-phase is not just a passive copying exercise. The cell actively manages traffic on its own genome to keep the replication program on track.
Checkpoint Surveillance and Fork Rescue
Errors during replication are common enough that cells maintain a dedicated surveillance system running throughout S-phase. The central player is a kinase called ATR, which monitors the health of replication forks. When a fork stalls, perhaps because it hits a damaged base, a tightly bound protein, or a region running low on nucleotide building blocks, stretches of single-stranded DNA accumulate and trigger ATR activation. ATR then signals through a downstream kinase called Chk1, which slows the overall pace of replication, suppresses the firing of new origins, and buys the cell time to fix the problem.17PubMed Central. ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage Inhibiting ATR experimentally causes DNA damage to pile up rapidly, underscoring how essential this checkpoint is even during normal replication.
ATR activation can also work locally, right at the site of a blocked fork, triggering rapid phosphorylation of histone H2AX in the vicinity and recruiting repair factors to the immediate area.18eLife. A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells Recent work has shown that the proteins RTF2 and CLASPIN physically position Chk1 at replication forks, keeping it poised for rapid activation. This fork-localized Chk1 activity helps maintain normal S-phase progression even when nothing is going wrong, functioning as a kind of baseline safety monitor that also enforces the checkpoint when cells transition from S into G2.19PubMed Central. RTF2 and CLASPIN localize CHK1 to the replication fork to control S-phase progression
When a fork does stall, the cell has backup polymerases that can step in. One of these, a translesion polymerase called Pol kappa, protects stalled forks from collapsing and helps restart DNA synthesis when nucleotide levels run low. This is a distinct role from its better-known job of copying past damaged bases; Pol kappa actively promotes recovery of stalled forks, not just lesion bypass.20PubMed Central. Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery
How Replication Wraps Up
Replication does not end all at once. Because forks travel outward from thousands of origins, termination happens wherever two converging forks meet. At that point, the last stretch of parental DNA between them is unwound, the leading strand of one fork is joined to the lagging strand of the other, and the replication helicase complex, called CMG, must be removed from the DNA. CMG unloading is not passive. The cell actively tags the Mcm7 subunit of CMG with chains of ubiquitin, which signals for the complex to be pulled off.21PubMed Central. Closing the MCM cycle at replication termination sites
This disassembly is carefully sequenced. CMG is only unloaded after the leading strand from one fork has been ligated to the lagging strand of the converging fork, meaning double-stranded DNA passes through the helicase’s central channel before the complex is released. In yeast, a specific ubiquitin ligase called SCF-Dia2 handles the ubiquitin tagging of Mcm7.22DASH. Regulation of CMG Helicase Disassembly During Replication Termination Premature helicase removal would leave gaps in the genome, so tying the unloading trigger to strand ligation is a safeguard against incomplete replication.
Fueling Replication With Nucleotides
Copying three billion base pairs of DNA in a few hours requires an enormous supply of the four nucleotide building blocks. The cell ramps up production of these molecules specifically during S-phase. A central enzyme in this supply chain is ribonucleotide reductase, or RNR, which converts ribonucleotides into the deoxyribonucleotides that DNA polymerases actually use. RNR expression rises during S-phase, driven in part by the transcription factor MYC. Another key enzyme, thymidylate synthase, provides the thymidine component of DNA and is also upregulated during this period.23PubMed Central. Regulation of mammalian nucleotide metabolism and biosynthesis – Section: Synthesis of the deoxyNTPs
This metabolic dependence is exploited by cancer drugs. The chemotherapy agent 5-fluorouracil, one of the oldest and most widely used anti-cancer drugs, works by blocking thymidylate synthase. Without this enzyme, the cell cannot produce thymidine monophosphate, a building block essential for DNA replication. The resulting depletion of thymidine triphosphate throws off the balance of all four nucleotide pools, severely disrupting DNA synthesis and ultimately killing rapidly dividing cells.24PubMed Central. 5-Fluorouracil: Mechanisms of Resistance and Reversal Strategies Other drugs target different nodes in the same supply chain; the common thread is that S-phase cells, with their voracious appetite for nucleotides, are uniquely vulnerable.
Measuring S-Phase in the Lab
Researchers studying S-phase often need to know how long it lasts and which cells are currently in it. The standard approach takes advantage of the fact that cells actively copying DNA will incorporate synthetic nucleotide analogs into their new strands. Historically, the analog of choice was BrdU, a modified form of thymidine. More recently, a second analog called EdU has become popular because it can be detected through a simple chemical reaction rather than requiring antibodies and harsh acid treatment. A dual-pulse method, in which cells are exposed first to EdU and then to BrdU at a defined interval, allows researchers to identify cells entering and exiting S-phase and calculate its duration.25PubMed Central. Measuring S-Phase Duration from Asynchronous Cells Using Dual EdU-BrdU Pulse-Chase Labeling Flow Cytometry26PubMed. Dual-pulse labeling using 5-ethynyl-2′-deoxyuridine (EdU) and 5-bromo-2′-deoxyuridine (BrdU) in flow cytometry These techniques have been essential for understanding how S-phase duration varies between cell types, speeds up or slows down in cancer, and responds to drug treatments.
S-Phase Without Cell Division
Not every S-phase leads to mitosis. Some cell types deliberately skip division after copying their DNA, a strategy called endoreplication. In fruit flies, many larval tissues use a streamlined cycle that alternates only between S-phase and a gap phase, skipping mitosis entirely. The result is polyploid cells, cells with multiple copies of the genome, which grow much larger than their diploid neighbors. This is not a glitch but a developmental strategy: the oversized cells serve structural or metabolic functions that smaller cells could not.27PubMed Central. Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells Mammals use endoreplication too, though in more limited contexts. Liver cells and certain placental cells in humans can become polyploid through variants of this process. These cases remind us that S-phase is not just a cog in the standard division machine; it is a flexible module that evolution has repurposed for different biological needs.