What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

DNA Replication Occurs in S Phase: Mechanisms and Evidence

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to the Cell Cycle and S Phase

The cell cycle is the ordered sequence of events by which a eukaryotic cell duplicates its contents and divides into two daughter cells. It is conventionally divided into four phases: G1 (gap 1), S (synthesis), G2 (gap 2), and M (mitosis). G1, S, and G2 collectively constitute interphase, the period between successive mitotic divisions. During G1, the cell grows, carries out its differentiated functions, and assesses both its internal state and the extracellular environment before committing to another round of division. The S phase, which follows G1, is the period during which the entire nuclear genome is duplicated. G2 follows S phase and provides a second gap period during which the cell verifies that replication has been completed accurately and that the DNA is undamaged before entering mitosis. The M phase encompasses mitosis (nuclear division) and cytokinesis (cytoplasmic division), producing two genetically identical daughter cells.

Overview of the Cell Cycle

The duration of each phase varies by organism and cell type. A typical mammalian cell in culture has a total cycle time of approximately 24 hours, with G1 lasting about 11 hours, S phase about 8 hours, G2 about 4 hours, and M phase about 1 hour. Rapidly dividing embryonic cells, such as those of Xenopus laevis (the African clawed frog), can complete S phase in as little as 20 minutes because they have already stockpiled all required replication proteins and lack G1 and G2 checkpoints. In contrast, slowly dividing cells such as adult hepatocytes may remain in G0 (a reversible quiescent state) for months or years before re-entering the cycle.

The progression through these phases is driven by the periodic activation of cyclin-dependent kinases (CDKs). CDKs are serine/threonine kinases that are constitutively expressed but only catalytically active when bound to a regulatory cyclin subunit. Different cyclins are synthesized and degraded at specific points in the cycle, providing temporal control over CDK activity. Cyclin D–CDK4/6 and cyclin E–CDK2 drive the G1/S transition; cyclin A–CDK2 is active during S phase; and cyclin B–CDK1 drives the G2/M transition. The precise regulation of CDK activity ensures that DNA replication occurs only during S phase and only once per cell cycle.

Defining S Phase

S phase is operationally defined as the interval during which DNA synthesis occurs. This definition was established experimentally using pulse-labeling with radioactive thymidine or, more commonly today, with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU), which is incorporated into newly synthesized DNA. When cells are exposed to a brief pulse of BrdU and then fixed and stained with an anti-BrdU antibody, only those cells that were actively replicating during the pulse show a positive signal. By combining this approach with flow cytometry, one can determine both the fraction of cells in S phase and the position of individual cells within S phase based on total DNA content (cells in early S have only slightly more DNA than G1 cells, while cells in late S approach the 4N DNA content of G2 cells).

The key point is that DNA replication occur in S phase and not in G1 or G2. This restriction is not accidental; it is the result of elaborate regulatory mechanisms that have evolved to ensure genome stability. Replicating DNA outside of S phase would be catastrophic, as it would lead to gene amplification, aneuploidy, and genomic instability—hallmarks of cancer.

The Central Dogma: DNA Replication Timing

The statement that DNA replication occurs only in S phase is a central tenet of molecular biology. The timing of replication is not merely a consequence of substrate availability; it is actively enforced by the cell cycle machinery. Understanding why this timing matters requires an appreciation of the consequences of unscheduled replication.

Why Timing Matters

There are several compelling reasons why DNA replication must be confined to S phase. First, replication must be coordinated with cell growth. A cell that replicated its DNA without having grown sufficiently would produce daughter cells that are smaller than normal, potentially compromising their viability. Second, replication must be completed before mitosis, because the condensed chromosomes that segregate during M phase must each contain exactly one copy of the genetic material. If replication were incomplete, sister chromatids would not form, and chromosome segregation would fail. Third, the cell must have time to repair any replication errors or DNA damage before the chromosomes are partitioned. The G2 phase provides this window.

Most fundamentally, however, the restriction of replication to S phase prevents re-replication. If a cell were to replicate its DNA twice in a single cycle, it would produce daughter cells with more than the normal complement of chromosomes. The mechanisms that prevent re-replication are collectively known as replication licensing control, and they are discussed in detail below.

Cell Cycle Checkpoints

The cell cycle is monitored by checkpoint pathways that halt progression when problems are detected. The G1/S checkpoint (also called the restriction point in mammalian cells) assesses cell size, nutrient availability, growth factor signaling, and DNA damage. If conditions are unfavorable, the cell exits the cycle into G0 or arrests in G1. The G2/M checkpoint verifies that DNA replication has been completed and that the DNA is undamaged before allowing entry into mitosis. The intra-S phase checkpoint monitors replication fork progression and responds to replication stress by slowing fork movement and stabilizing stalled forks.

These checkpoints are not merely passive monitors; they actively inhibit CDK activity. The G1/S checkpoint is enforced by the tumor suppressor protein p53, which is stabilized in response to DNA damage and induces transcription of the CDK inhibitor p21. The G2/M checkpoint is enforced by the kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related), which phosphorylate and inactivate the phosphatase Cdc25, thereby preventing activation of cyclin B–CDK1. The intra-S checkpoint is mediated by ATR, which is recruited to single-stranded DNA at stalled replication forks and phosphorylates downstream effectors such as Chk1.

Molecular Mechanisms Initiating Replication in S Phase

The initiation of DNA replication in S phase is a two-step process: origin licensing occurs in G1, and origin firing occurs in S phase. This temporal separation is essential for ensuring that each origin is used no more than once per cell cycle.

Origin Licensing

DNA replication begins at specific chromosomal locations called Replication Origins. In Saccharomyces cerevisiae (budding yeast), origins are defined by a conserved 11-base-pair consensus sequence called the autonomously replicating sequence (ARS) consensus sequence. In higher eukaryotes, origins are less well-defined by sequence and are instead specified by chromatin structure, DNA topology, and transcriptional activity. Regardless of the organism, the first step in origin activation is the binding of the origin recognition complex (ORC), a six-subunit ATPase complex, to the origin DNA.

ORC serves as a landing pad for the licensing factors Cdc6 and Cdt1, which in turn recruit the minichromosome maintenance (MCM) complex. The MCM complex is a hexamer of six related proteins (Mcm2–Mcm7) that forms the core of the replicative helicase. Loading of the MCM complex onto origin DNA requires ATP hydrolysis by ORC and Cdc6. The result is the pre-replicative complex (pre-RC), which marks origins as licensed for replication. Licensing can only occur during G1, when CDK activity is low. Once S phase begins and CDK activity rises, licensing is inhibited by multiple redundant mechanisms, including phosphorylation of Cdc6 (which targets it for ubiquitin-mediated degradation), nuclear export of Cdt1, and degradation of Cdt1 by the ubiquitin ligase SCF-Skp2.

Activation by CDKs

Origin firing in S phase requires the activity of S phase CDKs (cyclin E–CDK2 and cyclin A–CDK2) and the Dbf4-dependent kinase (DDK), which consists of the kinase Cdc7 bound to its regulatory subunit Dbf4. These kinases phosphorylate components of the pre-RC, including the MCM complex and the proteins Cdc45 and Sld3 (in yeast) or their functional homologs in higher eukaryotes (Treslin and TopBP1).

The phosphorylation events promote the recruitment of additional factors, including the helicase-activating protein GINS (Go-Ichi-Ni-San, named from the Japanese for 5-1-2-3, referring to the four subunits Sld5, Psf1, Psf2, and Psf3) and the polymerase-loading clamp PCNA (proliferating cell nuclear antigen). Together, the MCM complex, Cdc45, and GINS form the CMG complex, which is the active replicative helicase. The CMG complex unwinds the DNA duplex at the origin, creating a Replication Fork Bubble that expands bidirectionally as two replication forks move away from the origin.

Pre-replication Complex

The assembly of the pre-RC is a carefully ordered process that can be summarized as follows:

ORC binds to origin DNA in an ATP-dependent manner.
Cdc6 binds to the ORC–origin complex.
Cdt1, in complex with the MCM hexamer, is recruited to the origin.
ATP hydrolysis by ORC and Cdc6 drives the loading of the MCM double hexamer onto the DNA, encircling the duplex.
Cdt1 and Cdc6 are released, leaving the MCM complex stably bound as the pre-RC.

The loading of the MCM double hexamer is a key event because it establishes the bidirectional nature of replication. The two MCM hexamers are oriented head-to-head, and each will translocate in opposite directions along the DNA template during S phase. Importantly, the MCM complex is loaded in an inactive state; it only becomes an active helicase after phosphorylation by DDK and CDKs in S phase.

The Replication Fork: Machinery and Process

Once origins fire, the replication machinery assembles at each origin to form two replication forks that move in opposite directions. The Replication Fork Definition is the Y-shaped region where the parental DNA duplex is unwound and new DNA is synthesized. The fork is a dynamic assembly of proteins that must coordinate unwinding, priming, and polymerization with remarkable speed and accuracy.

Helicase Unwinding

The CMG complex is the replicative helicase in eukaryotes. It translocates along single-stranded DNA in the 3' to 5' direction, using the energy of ATP hydrolysis to unwind the duplex. As the helicase moves, it generates single-stranded DNA (ssDNA), which is immediately coated by replication protein A (RPA). RPA is a heterotrimeric ssDNA-binding protein that protects the exposed template from nucleases and prevents the formation of secondary structures that would impede the polymerases.

The unwinding reaction is processive: a single CMG complex can unwind tens of thousands of base pairs without dissociating. The rate of fork movement in mammalian cells is approximately 1–2 kilobases per minute, although this can vary depending on the cell type and the presence of replication stress.

Leading and Lagging Strand Synthesis

DNA polymerases are template-directed enzymes that synthesize DNA in the 5' to 3' direction. Because the two template strands at a replication fork are antiparallel, the two new strands must be synthesized differently. The leading strand is synthesized continuously in the same direction as fork movement, using the 3' to 5' template strand. The lagging strand is synthesized discontinuously in the opposite direction, using the 5' to 3' template strand, as a series of short fragments called Okazaki fragments.

The key enzymes at the fork are:

DNA polymerase α (Pol α): A polymerase with associated primase activity. It synthesizes short RNA–DNA primers (approximately 10 nucleotides of RNA followed by 20–30 nucleotides of DNA) at the origin and at the start of each Okazaki fragment.
DNA polymerase δ (Pol δ): The lagging strand polymerase. It extends Okazaki fragments after Pol α has laid down the primer.
DNA polymerase ε (Pol ε): The leading strand polymerase. It is a highly processive enzyme that synthesizes the leading strand continuously.
PCNA: A sliding clamp that encircles DNA and tethers the polymerases to their templates, dramatically increasing their processivity.
Replication factor C (RFC): A clamp loader that uses ATP hydrolysis to load PCNA onto primed DNA.
Flap endonuclease 1 (FEN1): An endonuclease that removes the RNA primers from Okazaki fragments.
DNA ligase I: The enzyme that seals the nick between adjacent Okazaki fragments.

The synthesis of the lagging strand is a complex choreography. Pol α synthesizes the RNA–DNA primer, then dissociates. RFC loads PCNA onto the primer–template junction, and Pol δ takes over, extending the fragment until it reaches the 5' end of the previous Okazaki fragment. The RNA primer of the previous fragment is displaced into a flap by the continued polymerization of Pol δ, and this flap is cleaved by FEN1. Finally, DNA ligase I seals the nick.

The overall process is summarized in the following table:

Protein	Function	Strand
CMG complex (Mcm2-7, Cdc45, GINS)	Replicative helicase; unwinds DNA	Both
RPA	Binds and stabilizes single-stranded DNA	Both
Pol α/primase	Synthesizes RNA–DNA primers	Both
Pol ε	Leading strand synthesis	Leading
Pol δ	Lagging strand synthesis	Lagging
PCNA	Sliding clamp; increases processivity	Both
RFC	Clamp loader	Both
FEN1	Removes RNA primers	Lagging
DNA ligase I	Seals nicks	Lagging

Experimental Evidence for S Phase Replication

The conclusion that DNA replication occur in S phase is supported by decades of experimental evidence. Two classic approaches—pulse-chase labeling and cell fusion experiments—were instrumental in establishing this fact.

Pulse-Chase with BrdU

The most direct evidence that DNA synthesis is confined to S phase comes from pulse-labeling experiments. In a typical experiment, asynchronously growing cells are exposed to a short pulse (5–30 minutes) of BrdU, a thymidine analog that is incorporated into newly synthesized DNA. The cells are then fixed, and the incorporated BrdU is detected using a fluorescently labeled anti-BrdU antibody. Simultaneously, the total DNA content of each cell is measured by staining with a DNA-binding dye such as propidium iodide. Flow cytometry can then be used to correlate BrdU incorporation with DNA content.

The results are unambiguous: only cells with DNA content intermediate between 2N (G1) and 4N (G2/M) incorporate BrdU. Cells in G1 and G2/M show no BrdU signal, demonstrating that no DNA synthesis occurs in these phases. This experiment can be refined by using a pulse-chase protocol, in which cells are labeled with BrdU and then chased with excess thymidine. By harvesting cells at various times after the chase and analyzing them by flow cytometry, one can track the progression of the labeled cohort through S phase and into G2 and M.

Cell Fusion Experiments

A second classic approach is cell fusion. In these experiments, cells in different phases of the cell cycle are fused using inactivated Sendai virus or polyethylene glycol, creating heterokaryons (cells with two or more nuclei). The behavior of each nucleus can then be monitored.

The key finding from these experiments was that when an S phase cell is fused with a G1 cell, the G1 nucleus is induced to enter S phase prematurely. This demonstrates that S phase cells contain diffusible factors (now known to be S phase CDKs) that promote replication. Conversely, when an S phase cell is fused with a G2 cell, the G2 nucleus does not initiate replication. This shows that the G2 nucleus has already completed replication and is refractory to S phase signals, consistent with the idea that replication is limited to one round per cycle.

These cell fusion experiments, performed by P.N. Rao and R.T. Johnson in the 1970s, were foundational in establishing the concept of replication licensing and the existence of positive and negative regulators of S phase entry.

Regulation and Checkpoints Ensuring Replication Completeness

The cell must not only initiate replication at the correct time but also ensure that replication is completed before mitosis. This is achieved through a combination of checkpoint surveillance and the intrinsic organization of replication origins.

Intra-S Phase Checkpoint

The intra-S phase checkpoint is a signaling pathway that monitors replication fork progression and responds to replication stress. Replication stress is defined as any condition that slows or stalls fork movement, including nucleotide depletion, DNA damage, and difficult-to-replicate genomic regions such as common fragile sites and telomeres.

The primary sensor of replication stress is ATR. When a replication fork stalls, the helicase continues to unwind DNA ahead of the polymerase, generating a stretch of single-stranded DNA. This ssDNA is rapidly coated by RPA, and the RPA–ssDNA complex recruits ATR via its interacting partner ATRIP. ATR then phosphorylates the effector kinase Chk1, which in turn phosphorylates and inhibits Cdc25A, a phosphatase required for CDK activation. The result is a slowing of origin firing and a stabilization of stalled forks, giving the cell time to resolve the problem.

The intra-S checkpoint also prevents the firing of late origins when early origins have stalled. In an unperturbed S phase, origins fire throughout the phase, with early-firing origins activated in early S and late-firing origins activated in late S. If replication stress is detected, the checkpoint suppresses late origin firing, reducing the load on the replication machinery and allowing the cell to focus its resources on completing replication from the already-active forks.

Replication Stress Response

When replication forks stall, they must be stabilized to prevent their collapse into double-strand breaks. The key player in fork stabilization is the protein BRCA1, which is recruited to stalled forks and helps to protect the nascent DNA strands from nucleolytic degradation. The Fanconi anemia pathway, which includes the proteins FANCD2 and FANCI, also plays a role in fork protection, particularly at interstrand crosslinks.

If a fork cannot be restarted, it may collapse, generating a one-ended double-strand break. This break is repaired by homologous recombination, a process that requires the recombinase RAD51. The repair of collapsed forks is essential for completing replication, and defects in this process are associated with cancer predisposition syndromes such as Fanconi anemia and hereditary breast and ovarian cancer (due to BRCA1/BRCA2 mutations).

The Replication Fork Stalling response is a complex and highly regulated process. It involves the recruitment of multiple repair and signaling proteins to the stalled fork, the transient inhibition of origin firing, and the eventual restart of the fork or its repair by recombination.

Common Misconceptions and Pitfalls

Students frequently encounter several misconceptions when learning about DNA replication timing. Addressing these directly will help clarify the material.

Misconception: Replication in G1

A common error is to assume that because G1 is the phase before S, some DNA synthesis must occur in G1. This is incorrect. G1 is a period of cell growth and preparation, but no DNA synthesis occurs. The G1/S checkpoint ensures that the cell only commits to replication when conditions are appropriate. The only DNA synthesis that occurs in G1 is mitochondrial DNA replication, which is independent of the nuclear cell cycle.

Misconception: Single Origin in Eukaryotes

Another common error is to assume that eukaryotic chromosomes, like bacterial chromosomes, have a single origin of replication. This is incorrect. Eukaryotic chromosomes are linear and much larger than bacterial chromosomes; a single origin would require an impossibly long time to replicate the entire chromosome. Instead, eukaryotic chromosomes contain hundreds to thousands of origins. For example, S. cerevisiae has approximately 400 origins distributed across its 16 chromosomes, while human cells have an estimated 30,000–50,000 origins. Only a subset of these origins fires in any given S phase; the rest remain dormant and serve as backup in case of replication stress.

Misconception: All Origins Fire Simultaneously

A related misconception is that all origins fire at the same time at the start of S phase. In reality, origin firing is staggered throughout S phase. Early-firing origins are located in euchromatin (open, transcriptionally active chromatin), while late-firing origins are located in heterochromatin (condensed, transcriptionally silent chromatin). The timing of origin firing is determined by the local chromatin environment and by the availability of limiting factors such as Cdc45 and DDK.

Misconception: Replication Is Continuous

Students sometimes think that once an origin fires, the replication fork moves continuously until it reaches the end of the chromosome or meets a fork from the adjacent origin. In reality, forks frequently pause or stall at difficult-to-replicate regions, and they can be restarted or bypassed. The intra-S checkpoint is specifically designed to deal with these obstacles.

Summary and Study Tips

Key Takeaways

DNA replication occur in S phase of the cell cycle, not in G1 or G2.
The timing of replication is enforced by CDK activity, which is low in G1 (allowing origin licensing) and high in S phase (triggering origin firing).
Origin licensing involves the assembly of the pre-RC (ORC, Cdc6, Cdt1, and MCM complex) during G1.
Origin firing in S phase requires CDK and DDK activity, leading to the formation of the CMG helicase and the assembly of the replication fork.
The leading strand is synthesized continuously by Pol ε, while the lagging strand is synthesized discontinuously as Okazaki fragments by Pol δ.
The intra-S phase checkpoint, mediated by ATR and Chk1, monitors fork progression and responds to replication stress.
Experimental evidence from BrdU pulse-labeling and cell fusion experiments demonstrates that replication is confined to S phase.

Exam Preparation

To prepare for exams, focus on understanding the logic of the system rather than memorizing isolated facts. Ask yourself the following questions:

Why is it important that replication occurs only once per cell cycle?
What are the molecular mechanisms that prevent re-replication?
How does the cell coordinate the firing of thousands of origins?
What happens when replication forks stall, and how does the cell respond?

Practice drawing the replication fork and labeling all the proteins involved. Be able to explain the difference between leading and lagging strand synthesis. Understand the experimental evidence for S phase replication, and be able to design a simple experiment to test whether a given drug affects replication timing.

Frequently Asked Questions

Does DNA replication occur in S phase?

Yes. DNA replication occur in S phase of the cell cycle. This is the only phase during which nuclear DNA is synthesized. The S phase is defined by this activity, and the cell cycle machinery ensures that replication is initiated only after the G1/S transition and is completed before the cell enters G2.

Why does DNA replication only happen in S phase?

DNA replication is restricted to S phase because the enzymes that initiate replication (CDKs and DDK) are only active during S phase, while the factors that license origins (Cdc6, Cdt1) are only present and active during G1. This temporal separation ensures that origins are licensed in G1 but only fire in S phase, and it prevents re-replication within a single cell cycle.

What happens if DNA replication occurs in G1?

If DNA replication were to occur in G1, it would lead to re-replication—the duplication of DNA that has already been replicated. This would result in gene amplification, DNA damage, and genomic instability. The cell has multiple redundant mechanisms to prevent this, including the degradation of licensing factors upon CDK activation and the nuclear export of Cdt1.

How do we know DNA replication occurs in S phase?

The most direct evidence comes from pulse-labeling experiments with thymidine analogs such as BrdU. Cells are exposed to a short pulse of BrdU, and the incorporated analog is detected by immunofluorescence or flow cytometry. Only cells in S phase, as determined by their DNA content, show BrdU incorporation. Cell fusion experiments, in which S phase cells are fused with G1 or G2 cells, provide additional evidence by showing that S phase cells contain diffusible factors that can induce replication in G1 nuclei but not in G2 nuclei.

Is DNA replication in S phase the same in prokaryotes?

No. Prokaryotes such as Escherichia coli do not have a cell cycle with distinct G1, S, and G2 phases. Instead, they replicate their DNA continuously during rapid growth, with multiple replication forks operating simultaneously on the circular chromosome. The regulation of replication initiation in prokaryotes is also different, involving the initiator protein DnaA and the origin oriC, rather than CDKs and licensing factors.

What is the S phase checkpoint?

The S phase checkpoint, also called the intra-S phase checkpoint, is a signaling pathway that monitors replication fork progression and responds to replication stress. It is mediated by the kinases ATR and Chk1, which slow origin firing, stabilize stalled forks, and prevent the cell from entering mitosis with incompletely replicated DNA.

Can DNA replication occur outside S phase?

Under normal conditions, no. Nuclear DNA replication is strictly confined to S phase. However, certain specialized processes involve DNA synthesis outside of S phase, including DNA repair synthesis (such as nucleotide excision repair and homologous recombination), which involves only short patches of DNA, and mitochondrial DNA replication, which occurs throughout the cell cycle.