Hubbry Logo
S phaseS phaseMain
Open search
S phase
Community hub
S phase
logo
8 pages, 0 posts
0 subscribers
Be the first to start a discussion here.
Be the first to start a discussion here.
S phase
S phase
S phase
View on Wikipedia
from Wikipedia
Asymmetry in the synthesis of leading and lagging strands

S phase (Synthesis phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase.[1] Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.

Regulation

[edit]
Main article: G1/S transition

Entry into S-phase is controlled by the G1 restriction point (R), which commits cells to the remainder of the cell-cycle if there is adequate nutrients and growth signaling.[2] This transition is essentially irreversible; after passing the restriction point, the cell will progress through S-phase even if environmental conditions become unfavorable.[2]

Accordingly, entry into S-phase is controlled by molecular pathways that facilitate a rapid, unidirectional shift in cell state. In yeast, for instance, cell growth induces accumulation of Cln3 cyclin, which complexes with the cyclin dependent kinase CDK2.[3] The Cln3-CDK2 complex promotes transcription of S-phase genes by inactivating the transcriptional repressor Whi5.[3] Since upregulation of S-phase genes drive further suppression of Whi5, this pathway creates a positive feedback loop that fully commits cells to S-phase gene expression.[3]

A remarkably similar regulatory scheme exists in mammalian cells.[3] Mitogenic signals received throughout G1-phase cause gradual accumulation of cyclin D, which complexes with CDK4/6.[3] Active cyclin D-CDK4/6 complex induces release of E2F transcription factor, which in turn initiates expression of S-phase genes.[3] Several E2F target genes promote further release of E2F, creating a positive feedback loop similar to the one found in yeast.[3]

DNA replication

[edit]
Main article: DNA replication
Steps in DNA synthesis

Throughout M phase and G1 phase, cells assemble inactive pre-replication complexes (pre-RC) on replication origins distributed throughout the genome.[4] During S-phase, the cell converts pre-RCs into active replication forks to initiate DNA replication.[4] This process depends on the kinase activity of Cdc7 and various S-phase CDKs, both of which are upregulated upon S-phase entry.[4]

Activation of the pre-RC is a closely regulated and highly sequential process. After Cdc7 and S-phase CDKs phosphorylate their respective substrates, a second set of replicative factors associate with the pre-RC.[4] Stable association encourages MCM helicase to unwind a small stretch of parental DNA into two strands of ssDNA, which in turn recruits replication protein A (RPA), an ssDNA binding protein.[4] RPA recruitment primes the replication fork for loading of replicative DNA polymerases and PCNA sliding clamps.[4] Loading of these factors completes the active replication fork and initiates synthesis of new DNA.

Complete replication fork assembly and activation only occurs on a small subset of replication origins. All eukaryotes possess many more replication origins than strictly needed during one cycle of DNA replication.[5] Redundant origins may increase the flexibility of DNA replication, allowing cells to control the rate of DNA synthesis and respond to replication stress.[5]

Histone synthesis

[edit]

Since new DNA must be packaged into nucleosomes to function properly, synthesis of canonical (non-variant) histone proteins occurs alongside DNA replication. During early S-phase, the cyclin E-Cdk2 complex phosphorylates NPAT, a nuclear coactivator of histone transcription.[6] NPAT is activated by phosphorylation and recruits the Tip60 chromatin remodeling complex to the promoters of histone genes.[6] Tip60 activity removes inhibitory chromatin structures and drives a three to ten-fold increase in transcription rate.[1][6]

In addition to increasing transcription of histone genes, S-phase entry also regulates histone production at the RNA level. Instead of polyadenylated tails, canonical histone transcripts possess a conserved 3` stem loop motif that selective binds to Stem Loop Binding Protein (SLBP).[7] SLBP binding is required for efficient processing, export, and translation of histone mRNAs, allowing it to function as a highly sensitive biochemical "switch".[7] During S-phase, accumulation of SLBP acts together with NPAT to drastically increase the efficiency of histone production.[7] However, once S-phase ends, both SLBP and bound RNA are rapidly degraded.[8] This immediately halts histone production and prevents a toxic buildup of free histones.[9]

Nucleosome replication

[edit]
Conservative reassembly of core H3/H4 nucleosome behind the replication fork.

Free histones produced by the cell during S-phase are rapidly incorporated into new nucleosomes. This process is closely tied to the replication fork, occurring immediately in "front" and "behind" the replication complex. Translocation of MCM helicase along the leading strand disrupts parental nucleosome octamers, resulting in the release of H3-H4 and H2A-H2B subunits.[10] Reassembly of nucleosomes behind the replication fork is mediated by chromatin assembly factors (CAFs) that are loosely associated with replication proteins.[4][11] Though not fully understood, the reassembly does not appear to utilize the semi-conservative scheme seen in DNA replication.[11] Labeling experiments indicate that nucleosome duplication is predominantly conservative.[11][10] The paternal H3-H4 core nucleosome remains completely segregated from newly synthesized H3-H4, resulting in the formation of nucleosomes that either contain exclusively old H3-H4 or exclusively new H3-H4.[10][11] "Old" and "new" histones are assigned to each daughter strand semi-randomly, resulting in equal division of regulatory modifications.[10]

Reestablishment of chromatin domains

[edit]

Immediately after division, each daughter chromatid only possesses half the epigenetic modifications present in the paternal chromatid.[10] The cell must use this partial set of instructions to re-establish functional chromatin domains before entering mitosis.

For large genomic regions, inheritance of old H3-H4 nucleosomes is sufficient for accurate re-establishment of chromatin domains.[10] Polycomb Repressive Complex 2 (PRC2) and several other histone-modifying complexes can "copy" modifications present on old histones onto new histones.[10] This process amplifies epigenetic marks and counters the dilutive effect of nucleosome duplication.[10]

However, for small domains approaching the size of individual genes, old nucleosomes are spread too thinly for accurate propagation of histone modifications.[10] In these regions, chromatin structure is probably controlled by incorporation of histone variants during nucleosome reassembly.[10] The close correlation seen between H3.3/H2A.Z and transcriptionally active regions lends support to this proposed mechanism.[10] Unfortunately, a causal relationship has yet to be proven.[10]

DNA damage checkpoints

[edit]

During S-phase, the cell continuously scrutinizes its genome for abnormalities. Detection of DNA damage induces activation of three canonical S-phase "checkpoint pathways" that delay or arrest further cell cycle progression:[12]

  1. The Replication Checkpoint detects stalled replication forks by integrating signals from RPA, ATR Interacting Protein (ATRIP), and RAD17.[12] Upon activation, the replication checkpoint upregulates nucleotide biosynthesis and blocks replication initiation from unfired origins.[12] Both of these processes contribute to rescue of stalled forks by increasing the availability of dNTPs.[12]
  2. The S-M Checkpoint blocks mitosis until the entire genome has been successfully duplicated.[12] This pathway induces arrest by inhibiting the Cyclin-B-CDK1 complex, which gradually accumulates throughout the cell cycle to promote mitotic entry.[12]
  3. The intra-S Phase Checkpoint detects Double Strand Breaks (DSBs) through activation of ATR and ATM kinases.[12] In addition to facilitating DNA repair, active ATR and ATM stalls cell cycle progression by promoting degradation of CDC25A, a phosphatase that removes inhibitory phosphate residues from CDKs.[12] Homologous recombination, an accurate process for repairing DNA double-strand breaks, is most active in S phase, declines in G2/M and is nearly absent in G1 phase.[13]

In addition to these canonical checkpoints, recent evidence suggests that abnormalities in histone supply and nucleosome assembly can also alter S-phase progression.[14] Depletion of free histones in Drosophila cells dramatically prolongs S-phase and causes permanent arrest in G2-phase.[14] This unique arrest phenotype is not associated with activation of canonical DNA damage pathways, indicating that nucleosome assembly and histone supply may be scrutinized by a novel S-phase checkpoint.[14]

See also

[edit]

References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

S phase

View on Grokipedia
from Grokipedia
The S phase, or synthesis phase, is a critical stage of the eukaryotic during which the cell's DNA is precisely duplicated to produce two identical copies of each , ensuring genetic continuity for daughter cells upon division. This replication occurs semi-conservatively, transforming the DNA content from 2n to 4n, and is confined to , following the and preceding the . In typical mammalian cells, the S phase duration ranges from 8 to 12 hours, comprising roughly half of the total ~24-hour in rapidly dividing cells. The primary process in S phase involves the activation of thousands of replication origins across the , where the (ORC) and (MCM) proteins initiate bidirectional DNA synthesis by DNA polymerases, resulting in the formation of tightly associated . Centrosomes and centrioles (in cells) also duplicate during this phase to support subsequent . Progression into S phase is gated by the G1/S checkpoint, often called the , which integrates extracellular growth signals and internal assessments of DNA integrity before committing to replication. S phase entry and execution are tightly regulated by cyclin-dependent kinases (CDKs), particularly CDK2 complexed with , which phosphorylate targets to replication origins in G1 and trigger firing in S phase, while ligases and checkpoints prevent over-replication or proceed in the face of damage. damage detected during S phase activates repair pathways, such as those involving , which can halt progression or induce arrest to maintain genomic stability. Dysregulation of S phase, as seen in cancer, can lead to genomic instability, highlighting its role in both normal proliferation and .

Overview

Definition and role

The S phase, or synthesis phase, is the second stage of the eukaryotic cell cycle, occurring after the G1 phase and before the G2 phase, during which the cell's entire genome undergoes semiconservative replication to produce two identical copies of each DNA molecule. This process doubles the DNA content from 2C to 4C, preparing the cell for subsequent division while maintaining genetic stability. The primary role of S phase is to ensure genetic continuity across generations by duplicating the genome exactly once per cell cycle, thereby preventing catastrophic errors such as mutations, gene amplification, or aneuploidy that could arise from incomplete or over-replication. This duplication is crucial for the equitable segregation of sister chromatids during mitosis, where the replicated chromosomes are distributed to daughter cells. S phase is temporally coordinated with the preceding G1 phase, which assesses cellular readiness, and the following G2 phase, which verifies replication fidelity before mitosis. The existence of distinct phases, including S phase, was first established in the through pioneering radiolabeling experiments using on root tip cells of , conducted by Alma Howard and Stephen Pelc. These studies revealed a discrete period of intervening between and a subsequent postsynthetic gap, laying the foundation for understanding the ordered progression of eukaryotic . In somatic cells, S phase typically lasts 8–10 hours and involves replicating approximately 6 billion base pairs of diploid DNA at a rate of approximately 20–50 base pairs per second per replication fork, facilitated by thousands of origins of replication firing in a coordinated manner.

Timing and coordination with cell cycle

The S phase occurs immediately following the and precedes the in the eukaryotic , during which ensures duplication prior to . Its temporal placement is tightly coordinated to maintain genomic integrity, with the phase's onset triggered by the successful completion of G1 growth and readiness checks. In mammalian somatic cells, S phase typically lasts 8–10 hours, while in rapidly dividing early embryos, it is dramatically shorter, completing in approximately 3–4 minutes due to high replication fork speeds and dense origin firing. This variability in duration across organisms and cell types is largely influenced by the density of replication origins, with higher densities enabling faster coverage of the and shorter overall S phase times. Integration with ensures orderly progression, beginning with the in late G1, where cells commit irreversibly to entering S phase upon receiving sufficient growth signals and achieving a critical threshold. This commitment prevents reversal even if external stimuli are withdrawn, linking G1 competence to S phase initiation. At the downstream end, coordination with the G2/M transition is enforced by the checkpoint, which halts mitotic entry if replication remains incomplete, thereby blocking progression until all genomic regions are duplicated and damage is resolved. These checkpoints collectively synchronize S phase with the broader cycle, averting errors like . The timing of S phase onset and exit is governed by cell cycle oscillators, particularly the periodic fluctuations in levels, which rise to promote entry into and decline to facilitate progression to G2. These oscillations create a temporal framework that aligns replication with other cycle events, ensuring S phase neither overlaps with nor lags behind preparatory phases. Experimental quantification of S phase dynamics often employs combined with thymidine analogs like BrdU, which incorporate into newly synthesized DNA, allowing real-time tracking of progression through of DNA content and analog incorporation. This approach reveals cell-to-cell variability and average durations, providing insights into mechanisms without disrupting cycle flow.

Regulation

Molecular triggers for entry

The transition into S phase is gated by the in late G1, an irreversible commitment to progression that depends on sufficient growth factors and nutrient availability to ensure cellular resources for . This checkpoint integrates extracellular signals, such as mitogens, which activate signaling pathways leading to the accumulation of D-type cyclins and their associated cyclin-dependent kinases (CDKs), thereby promoting passage through the . Once crossed, cells become independent of external stimuli and proceed to prepare for . A critical preparatory step is the assembly of the (pre-RC) during , which licenses replication origins for subsequent activation. The (ORC) binds to specific DNA sequences at potential origins, recruiting Cdc6 and Cdt1 to load the MCM2-7 as a double hexamer, forming the pre-RC. This licensing is tightly regulated to occur only in G1; in S, G2, and M phases, mechanisms including CDK-mediated of Cdc6, Cdt1 degradation, and geminin binding to Cdt1 prevent re-licensing, ensuring genome duplication happens once per cycle. Entry into S phase is further triggered by the activation of transcription factors, which drive expression of genes essential for . In early G1, the (Rb) binds and represses ; however, sequential phosphorylation of Rb by D-CDK4/6 and then E-CDK2 complexes releases , allowing it to transcribe S-phase genes such as those encoding and . This transcriptional program, in coordination with pre-RC licensing, initiates origin firing at designated sites, with mammalian cells activating approximately 30,000 to 50,000 origins to replicate the .

Control by cyclins and kinases

The progression of S phase is primarily orchestrated by cyclin-dependent kinases (CDKs), which form complexes with specific cyclins to drive DNA replication initiation and elongation while preventing aberrant re-replication. These enzymes phosphorylate key substrates, including components of the and transcription factors, ensuring timely and controlled . A pivotal complex is cyclin E bound to CDK2, which peaks in activity during late G1 and early S phase to trigger origin firing. This complex phosphorylates the (Rb), leading to the release of transcription factors that activate genes essential for S phase entry, such as those encoding and . Additionally, cyclin E-CDK2 phosphorylates CDC6, facilitating the loading of the MCM onto origins and promoting the onset of . The activity of this complex is modulated by cyclin E levels and CDK2's phosphorylation state, where activating on threonine 160 by CAK enhances kinase function; mathematically, CDK2 activity can be approximated as proportional to the product of cyclin E concentration and its phosphorylated state: CDK2 activity∝[Cyclin E]×(phosphorylation state)\text{CDK2 activity} \propto [\text{Cyclin E}] \times (\text{phosphorylation state}) This relationship underscores the tight regulation required for precise S phase initiation. Following entry, cyclin A-CDK2 sustains replication fork progression throughout S phase and enforces a barrier against re-replication. Cyclin A associates with CDK2 to phosphorylate substrates like CDC6, exporting it from the nucleus and inhibiting new assembly at licensed origins. This phosphorylation also targets Cdt1, a licensing factor, rendering it incapable of reloading MCM complexes and thus preventing DNA from being replicated more than once per cycle. By maintaining high activity during S phase, cyclin A-CDK2 ensures unidirectional progression of replication forks without untimely origin reactivation. To safeguard against uncontrolled entry, inhibitory mechanisms counteract CDK activity, particularly under cellular stress. CDK inhibitors such as p21 (CDKN1A) and p27 (CDKN1B) bind to cyclin E-CDK2 and cyclin A-CDK2 complexes, blocking their kinase activity and halting S phase progression in response to DNA damage or nutrient deprivation. For instance, p21 and p27 levels rise during stress, sequestering cyclins and preventing phosphorylation of pro-replicative targets. Complementing this, geminin accumulates in early S phase to sequester Cdt1, further inhibiting pre-replication complex reformation independently of direct CDK inhibition. This cyclin-CDK framework is highly conserved across eukaryotes, with variations reflecting species-specific adaptations. In budding yeast (), the S phase-promoting cyclins Clb5 and Clb6 pair with Cdk1 (the ortholog of metazoan CDK2) to drive origin firing and replication progression, mirroring the roles of cyclin E/A-CDK2 in mammals. Deletion of CLB5 delays S phase onset, while Clb6 provides partial redundancy, highlighting their functional equivalence. Despite these differences in cyclin nomenclature, the core mechanism—cyclin oscillation driving CDK-mediated for S phase control—remains invariant from to humans, ensuring genomic stability.

DNA replication process

Initiation of replication

The initiation of DNA replication begins with the recognition of replication origins by the (ORC), a heterohexameric protein that binds specifically to AT-rich DNA sequences in eukaryotic genomes. In budding , ORC preferentially associates with autonomously replicating sequences (ARS) characterized by an AT-rich composition, which facilitates initial DNA unwinding and serves as a platform for subsequent protein recruitment. Once bound, ORC recruits the Cdc6 ATPase and Cdt1 proteins during the of the , enabling the loading of the MCM2-7 complex onto double-stranded DNA in a head-to-head orientation, forming the pre-replicative complex (pre-RC). This encircles the DNA without unwinding it, positioning two MCM2-7 hexamers for bidirectional replication potential. Upon entry into S phase, activation of the pre-RC occurs through phosphorylation by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), which remodel the MCM2-7 complex and promote the recruitment of additional factors. DDK primarily phosphorylates MCM2-7 subunits, facilitating the loading of Cdc45 and the GINS complex to assemble the active CMG (Cdc45-MCM2-7-GINS) helicase, which encircles and unwinds the DNA at the origin. As the helicase progresses, replication protein A (RPA) binds to the exposed single-stranded DNA (ssDNA), stabilizing it and preventing secondary structure formation to support downstream priming and synthesis. This unwinding generates a bubble of ssDNA, marking the transition from licensing to active replication fork establishment. Primer synthesis is initiated by the DNA polymerase α-primase complex, which synthesizes short RNA-DNA hybrid primers on the exposed ssDNA templates. within the complex first polymerizes approximately 10 of RNA using NTP substrates, followed by extension with about 20 of DNA by the polymerase α subunit, creating a chimeric primer of roughly 30 total. These primers provide the 3'-OH end required for subsequent elongation by replicative polymerases. On the leading strand, ε binds and begins continuous synthesis from the primer in the 5' to 3' direction, while on the lagging strand, δ initiates discontinuous Okazaki fragment synthesis from additional primers generated periodically. The initial rate of replication fork progression is coordinated by the CMG helicase unwinding rate and polymerase synthesis, typically 1-2 kb/min (≈17-33 bp/s) in mammalian cells.

Elongation and fork progression

During the elongation phase of DNA replication, the leading strand is synthesized continuously in the 5' to 3' direction by DNA polymerase ε (Pol ε), which advances in tandem with the CMG helicase complex to replicate the parental template ahead of the fork. This process allows Pol ε to incorporate nucleotides without frequent dissociation, achieving high processivity of up to approximately 100 kb per binding event in eukaryotes. The lagging strand, in contrast, is replicated discontinuously by DNA polymerase δ (Pol δ) in short segments known as Okazaki fragments, each typically 100-200 nucleotides long in human cells. Pol δ synthesizes these fragments in the opposite direction to fork progression, initiating from RNA primers laid down by Pol α-primase. Maturation of on the lagging strand involves the coordinated removal of primers and gap filling to enable seamless ligation. RNase H specifically cleaves the -DNA hybrid at the 5' end of each fragment, while flap endonuclease 1 (FEN1) excises the remaining ribonucleotides through endonucleolytic cleavage of displaced flaps generated by Pol δ's strand displacement activity. The resulting nicks are then sealed by I, which catalyzes formation in an ATP-dependent manner, completing the lagging strand synthesis. Replication fork progression relies on accessory proteins that maintain replisome stability and coordinate unwinding with synthesis. The fork protection complex (FPC), comprising Timeless, Tipin, and Claspin, associates with the CMG and polymerases to prevent fork collapse and ensure coupling between helicase unwinding and . In eukaryotes, the CMG unwinds at a rate of approximately 50 base pairs per second, enabling bidirectional fork movement from each origin. Fork progression can be impeded by obstacles such as encounters with the transcription machinery or nucleosomes, which temporarily slow the and increase the risk of stalling. Transcription-replication conflicts arise when complexes block fork advance, particularly in highly transcribed regions, leading to reduced fork speeds. Similarly, nucleosomes ahead of the fork resist unwinding, and insufficient histone supply or assembly behind the fork further decelerates progression by disrupting dynamics. The overall time required for S phase completion can be estimated using the relation between genome size, fork speed, and the number of active origins, as replication proceeds bidirectionally from multiple sites: t=genome size2×fork speed×number of originst = \frac{\text{genome size}}{2 \times \text{fork speed} \times \text{number of origins}} This formula highlights how increased origin firing or faster forks reduce replication duration, ensuring timely genome duplication.

Termination and completion

As replication forks from adjacent origins converge during the final stages of S phase in eukaryotes, termination occurs without dedicated terminus sites, unlike in prokaryotes where specific ter sequences direct bidirectional fork meeting. Instead, eukaryotic forks merge stochastically across the genome, leading to the formation of catenated daughter DNA molecules due to unreleased intertwinings from replication. Decatenation is primarily mediated by topoisomerase II, which resolves these catenanes by introducing transient double-strand breaks and strand passage, ensuring the segregation of newly replicated chromosomes in the subsequent G2 and M phases. Any unreplicated gaps remaining after fork convergence, often arising from DNA lesions or replication stress that stalled forks earlier, are typically filled through post-replicative mechanisms such as translesion synthesis (TLS). TLS employs specialized low-fidelity s, including DNA ζ (Pol ζ), which acts as an extender to complete synthesis across damaged templates after an initial lesion-bypassing inserts opposite the damage. This process minimizes persistent single-stranded DNA gaps that could otherwise trigger genomic instability, although it introduces a higher risk compared to replicative s. Completion of S phase is marked by signals that halt further origin firing, including the deactivation of late-firing and dormant origins through rising (CDK) activity that and inhibits components like Cdc6 and Cdt1, preventing relicensing. Concurrently, undergoes decondensation facilitated by CDK2-mediated , which loosens higher-order structures and signals readiness for G2 entry by promoting nuclear reorganization and appropriate for the post-replicative state. The TRESLIN-MTBP complex further couples replication completion to G2/M transition by inhibiting premature progression until synthesis is fully resolved. The overall fidelity of eukaryotic DNA replication achieves an error rate of approximately 1 mistake per 10^9 to 10^10 bases incorporated, primarily through the combined actions of 3'→5' exonuclease proofreading by replicative polymerases (which corrects ~99% of initial mismatches) and post-replicative mismatch repair (MMR) systems that excise and replace erroneous segments. This multi-layered error correction ensures genome stability despite the immense scale of replication, with human cells duplicating ~6 × 10^9 base pairs per S phase.

Biosynthetic accompaniments

Histone synthesis and supply

During S phase, the canonical core histones H2A, H2B, H3, and H4 are synthesized in large quantities to package newly replicated DNA into chromatin, while replication-dependent variants of the linker histone H1 are also produced primarily during this period. These histones are encoded by gene clusters that lack introns and polyadenylation signals, ensuring their expression is tightly coupled to DNA replication. In mammalian cells, this process demands the production of approximately 400 million core histone molecules per cell division to form the required number of nucleosomes for the duplicated genome. Transcriptional activation of these histone genes occurs specifically at the through S-phase promoters that respond to signals. The key regulators include the HiNF-P ( nuclear factor P), which binds to these promoters, and NPAT (nuclear protein ataxia-telangiectasia locus), which is phosphorylated by E-CDK2 to recruit HiNF-P and initiate a cascade of coactivators within histone locus bodies. This upregulation ensures a burst of mRNA synthesis that peaks in early to mid-S phase. mRNAs are stabilized by a conserved 3' stem-loop structure bound by stem-loop binding protein (SLBP), which protects them from degradation and coordinates their processing and export during active replication. Excess s produced during S phase are temporarily stored in the nucleus by acidic chaperones to prevent and maintain supply. In mammalian cells, the chaperone NASP (nuclear acidic protein) forms reservoirs of soluble H3-H4 dimers, buffering fluctuations in histone demand. For delivery to replication sites, ASF1 acts as an intermediary chaperone that binds newly synthesized H3-H4 and transfers them to CAF-1 ( assembly factor 1), which is recruited to replication forks via PCNA to facilitate timely deposition. Following S phase completion, histone mRNAs are rapidly degraded through oligouridylation of the 3' stem-loop, , and exonucleolytic decay, ensuring synthesis ceases and excess proteins are cleared.

Nucleosome replication and assembly

During nucleosome replication in S phase, parental nucleosomes ahead of the replication fork are disassembled to allow progression, with existing recycled onto daughter DNA strands. Parental (H3-H4)2 tetramers are recycled symmetrically to both daughter DNA strands behind the replication fork. The N-terminal histone-binding domain of Mcm2 within the MCM2-7 complex binds and chaperones these parental tetramers, countering a weak leading-strand bias to ensure even distribution. This recycling ensures efficient reuse of pre-existing , preventing depletion and maintaining structure during replication. The process is tightly coupled to fork movement, with Mcm2 facilitating histone handover without requiring additional chaperones in some contexts. Newly synthesized histones, supplied in coordination with DNA replication to double chromatin content, are deposited onto the nascent strands to complement recycled parental histones. The chromatin assembly factor 1 (CAF-1) complex plays a central role, binding to (PCNA) at replication forks and depositing (H3-H4)2 tetramers onto newly synthesized DNA. This PCNA-dependent mechanism targets histone deposition precisely to sites of , ensuring balanced nucleosome formation on both leading and lagging strands. Following tetramer placement, H2A-H2B dimers are incorporated by chaperones such as NAP1 or FACT to complete octamer assembly. Nucleosome assembly proceeds in a stepwise manner: first, the (H3-H4)2 tetramer binds to DNA, wrapping approximately 120 base pairs around it, followed by the addition of two H2A-H2B dimers to form the full that encompasses about 147 base pairs of DNA. This sequential pathway maintains integrity and spacing, with one typically assembled per 147-200 base pairs to achieve proper compaction. The stoichiometry reflects the core particle structure, where the stabilizes the DNA wrap through electrostatic interactions. Replication-coupled assembly specifically incorporates the canonical histone variant H3.1, distinguishing it from replication-independent variants like H3.3. H3.1 is chaperoned by CAF-1 for deposition during S phase, ensuring that newly formed nucleosomes contain this variant on nascent DNA. This variant-specific integration supports the structural fidelity of replicated , with H3.1's unique sequence features aiding in stable octamer formation.

Chromatin maintenance

Reestablishment of epigenetic marks

During the S phase of the , DNA disrupts epigenetic marks on histones and DNA, necessitating their precise reestablishment to maintain patterns and cellular identity. Parental histones, carrying post-translational modifications (PTMs) such as , are semi-conservatively recycled to daughter strands, resulting in a twofold dilution of these marks due to the incorporation of newly synthesized, unmodified histones. This dilution is counteracted by active restoration mechanisms, where enzymes recognize residual parental marks and apply PTMs to new histones, ensuring epigenetic fidelity across generations. Histone modification inheritance primarily occurs through the symmetric segregation of modified parental to both leading and lagging daughter strands, facilitated by components of the like MCM2 and the POLE3/POLE4 subunits of . For repressive marks like , which defines pericentric , parental histones are preferentially transferred to leading strands at certain loci, such as LINE retrotransposons, via interactions between complex and polymerase epsilon. Active restoration involves methyltransferases like SETDB1, which, in complex with HP1α and assembly factor 1 (CAF1), monomethylates H3K9 (H3K9me1) on newly deposited histones during mid-S phase, providing a substrate for further trimethylation by SUV39H1/H2 to rebuild domains. Similarly, levels, associated with Polycomb-mediated , drop during S phase but are restored post-replication through mechanisms including delayed accumulation on nascent DNA, which is essential for proper , and facilitation by linker , which compacts to aid PRC2-mediated trimethylation, thereby preventing loss of memory. These processes exhibit two propagation modes: rapid restoration for some PTMs within one cell cycle via continuous modification of both old and new histones, and slower, multi-generational establishment for marks like and . DNA methylation patterns are reestablished post-replication through maintenance and de novo mechanisms. The primary maintenance methyltransferase preferentially methylates hemimethylated CpG sites generated during replication, ensuring propagation of parental methylation to daughter strands; this process is coordinated with S-phase progression via recruitment by UHRF1, which ubiquitylates and the replication factor PAF15 to tether to replication foci. De novo methylation, primarily catalyzed by DNMT3A and DNMT3B, occurs at unmethylated CpGs and contributes to establishing new patterns, particularly at imprinted loci or during developmental , though it overlaps with maintenance functions in replicating cells. also regulates the timing and targets of DNMT3 activity, linking maintenance to de novo events in an enzyme-dependent manner. Fidelity of epigenetic reestablishment relies on parental strand guidance, where recycled modified s serve as templates for enzymes to restore patterns on new . An information-theoretic model describes this as a threshold-filling , where methyltransferases fill short gaps (3-6 unmodified ) in daughter strands based on adjacent parental marks, minimizing inheritance errors to below 5% for marks like H3K27me3. Replication stress or disruptions in , such as asymmetric segregation, compromise this guidance, leading to loss of silencing and increased genomic instability. assembly, occurring concurrently with replication, provides the scaffold prerequisite for these restoration events.

Higher-order chromatin domains

During S phase, replication timing is tightly correlated with chromatin domain types, with regions typically replicating early and regions replicating late. This temporal program ensures that open, transcriptionally active euchromatic domains are duplicated first, while compact, repressive heterochromatic domains are replicated later, maintaining their distinct architectural identities. Following replication, higher-order chromatin loops are rapidly reformed through the reloading of and at anchor sites, which stabilize topologically associating domains (TADs). extrudes DNA loops until encountering CTCF-bound boundaries, thereby reanchoring TAD structures; this process is essential for restoring intra-domain interactions disrupted by replication forks. analyses reveal that TAD insulation, which weakens during S phase in a replication-timing-dependent manner (early for euchromatin-associated TADs, later for ), but recovers in , with insulation reaching a maximum as inter-TAD contacts are reestablished. A/B compartments, the large-scale spatial segregation of (A) and (B), are also reestablished shortly after replication, guided in part by epigenetic cues. In B compartments, HP1 binding to H3K9me3-marked nucleosomes facilitates rapid compaction and behind the , preventing dilution of repressive states during duplication. Insulator elements, primarily CTCF-bound sites, act as barriers to prevent the spreading of domains across TAD boundaries, ensuring faithful inheritance of architectural features. Recent studies indicate that promoter-proximal pausing of contributes to this barrier function by stabilizing local states and limiting ectopic interactions post-replication, thereby reinforcing domain insulation.

Checkpoints and responses

Intra-S phase checkpoint

The intra-S phase checkpoint serves as a critical surveillance mechanism that detects replication stress during S phase and modulates DNA replication to maintain genomic stability. Upon encountering replication obstacles, such as stalled replication forks, single-stranded DNA (ssDNA) regions become coated with replication protein A (RPA). This RPA-ssDNA complex recruits the ATR-ATRIP kinase complex, which senses the stress signal and initiates checkpoint activation. Subsequently, ATR recruits TopBP1, an activator that enhances ATR kinase activity, and the Rad17-RFC complex, which loads the 9-1-1 checkpoint clamp onto the DNA to further amplify the signal. This activation cascade ensures rapid response to fork stalling without immediately halting all cellular processes. Downstream of ATR activation, the checkpoint exerts multiple effects to mitigate replication stress. ATR phosphorylates the effector kinase Chk1, which in turn inhibits the firing of new replication origins by preventing the loading of additional replication factors, thereby conserving resources for ongoing forks. Additionally, Chk1 promotes the stabilization of stalled forks by counteracting nucleolytic degradation and excessive remodeling, reducing the risk of fork collapse into double-strand breaks. These actions collectively slow replication progression while allowing limited fork advancement under moderate stress. In physiological contexts, the intra-S phase checkpoint plays a vital role in preventing under-replication of the during conditions like hypoxia or depletion, which impair fork progression and dNTP availability. The core signaling pathway involves ATR-mediated of Chk1, leading to the ubiquitination and degradation of Cdc25A phosphatase, which inhibits CDK2 activity and further restricts origin firing and advancement. This mechanism ensures that replication completes faithfully even under resource-limited environments, coordinating with G2/M checkpoint activation to delay until S phase is resolved. Unlike the G1/S checkpoint, which primarily prevents entry into S phase in response to damage, the intra-S phase checkpoint permits partial replication progression while selectively suppressing late-origin firing and stabilizing existing forks, thereby integrating with broader arrest in G2. This distinction allows cells to adapt dynamically to ongoing stress without fully aborting the replication program.

DNA damage detection and repair

During S phase, DNA replication forks encounter various lesions that threaten genomic integrity, including UV-induced and double-strand breaks (DSBs) arising from replication fork collapse. UV-induced , such as cyclobutane pyrimidine dimers, distort the DNA helix and block replicative polymerases, while fork collapse often generates one-ended DSBs due to encounters with unresolved structures or exogenous agents. These lesions are detected by specialized sensors: the Mre11-Rad50-Nbs1 (MRN) complex rapidly binds DSB ends at collapsed forks, initiating processing and signaling through its and activities, whereas poly(ADP-ribose) polymerases (PARPs), particularly , detect single-strand breaks and other lesions by binding directly to DNA ends and catalyzing PARylation to recruit repair factors. Repair of these lesions during S phase primarily involves (NER) for bulky adducts like and (HR) for DSBs. NER, which operates efficiently in S phase under ATR kinase regulation, excises oligonucleotides containing the lesion via dual incisions on either side, allowing gap filling by replicative polymerases; global genome NER predominates during replication to handle replication-blocking damage. HR predominates for DSB repair in S and G2 phases, utilizing the newly synthesized sister as a template to restore sequence fidelity through strand invasion and , mediated by proteins like RAD51; this pathway is preferred over due to the availability of the homologous template. Checkpoint activation may transiently slow fork progression to facilitate these repairs. To coordinate repair with ongoing replication, cells employ translesion synthesis (TLS) polymerases, such as the Y-family Pol η and the B-family Pol ζ, which temporarily bypass lesions by incorporating opposite damaged bases, allowing fork progression while leaving gaps for later resolution; TLS is tightly regulated by ubiquitination of PCNA to switch polymerases. Post-replicative mismatch repair (MMR) then addresses replication errors or small loops in the nascent strand, recognizing mismatches via MSH2-MSH6 and excising the error-prone segment using nicks in the daughter strand as entry points. Failure to repair these lesions can lead to persistent DSBs or gaps, triggering through p53-dependent pathways or via sustained DNA damage response signaling. Recent studies using CRISPR-Cas9 to induce targeted damage have demonstrated enhanced HR efficiency specifically in S phase, with synchronization strategies boosting rates up to twofold by optimizing sister chromatid availability.

References

  1. https://www.ncbi.nlm.nih.gov/books/NBK26869/
  2. https://www.ncbi.nlm.nih.gov/books/NBK9876/
  3. https://open.lib.umn.edu/humanbiology/chapter/6-1-the-cell-cycle/
  4. https://pmc.ncbi.nlm.nih.gov/articles/PMC6496723/
  5. https://pmc.ncbi.nlm.nih.gov/articles/PMC6817399/
  6. https://www.ncbi.nlm.nih.gov/books/NBK26826/
  7. https://pmc.ncbi.nlm.nih.gov/articles/PMC9139170/
  8. https://www.ncbi.nlm.nih.gov/books/NBK9907/
  9. https://pmc.ncbi.nlm.nih.gov/articles/PMC1949372/
  10. https://genesdev.cshlp.org/content/30/15/1683.full
  11. https://www.sciencedirect.com/science/article/pii/S1097276517309693
  12. https://genesdev.cshlp.org/content/8/6/652
  13. https://royalsocietypublishing.org/doi/10.1098/rstb.2011.0082
  14. https://academic.oup.com/nar/article/29/7/1433/1001910
  15. https://www.nature.com/articles/7290031
  16. https://www.cell.com/molecular-cell/pdf/S1097-2765%2820%2930605-5.pdf
  17. https://www.nature.com/articles/s41467-021-24199-1
  18. https://www.nature.com/articles/nature11920.pdf
  19. https://www.cell.com/fulltext/S0092-8674%2805%2901231-6
  20. https://pmc.ncbi.nlm.nih.gov/articles/PMC3783049/
  21. https://www.nature.com/articles/s41586-022-04798-8
  22. https://pmc.ncbi.nlm.nih.gov/articles/PMC10909489/
  23. https://www.nature.com/articles/s41392-024-02080-z
  24. https://www.embopress.org/doi/10.1093/emboj/18.2.396
  25. https://www.sciencedirect.com/science/article/pii/S0960982295002521
  26. https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2021.774845/full
  27. https://www.nature.com/articles/s41467-021-27124-8
  28. https://www.pnas.org/doi/10.1073/pnas.0905281106
  29. https://genesdev.cshlp.org/content/6/9/1695
  30. https://pmc.ncbi.nlm.nih.gov/articles/PMC85702/
  31. https://pmc.ncbi.nlm.nih.gov/articles/PMC9898300/
  32. https://genesdev.cshlp.org/content/31/11/1073.long
  33. https://pmc.ncbi.nlm.nih.gov/articles/PMC3839609/
  34. https://pmc.ncbi.nlm.nih.gov/articles/PMC6847044/
  35. https://www.nature.com/articles/s41586-022-04829-4
  36. https://www.nature.com/articles/s41467-023-39441-1
  37. https://pmc.ncbi.nlm.nih.gov/articles/PMC3628110/
  38. https://elifesciences.org/articles/04988
  39. https://pmc.ncbi.nlm.nih.gov/articles/PMC5222725/
  40. https://pmc.ncbi.nlm.nih.gov/articles/PMC6386472/
  41. https://academic.oup.com/nar/article/47/16/8348/5549710
  42. https://www.sciencedirect.com/science/article/pii/S1097276522007596
  43. https://pmc.ncbi.nlm.nih.gov/articles/PMC2171796/
  44. https://pmc.ncbi.nlm.nih.gov/articles/PMC3639319/
  45. https://www.pnas.org/doi/10.1073/pnas.2100178118
  46. https://epigeneticsandchromatin.biomedcentral.com/articles/10.1186/s13072-022-00467-2
  47. https://www.pnas.org/doi/10.1073/pnas.0905651106
  48. https://genesdev.cshlp.org/content/22/1/50.full.html
  49. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010682
  50. https://www.sciencedirect.com/science/article/pii/S1097276504008020
  51. https://www.science.org/doi/10.1126/science.aau0294
  52. https://www.nature.com/articles/s41588-023-01476-x
  53. https://genesdev.cshlp.org/content/29/6/585
  54. https://pmc.ncbi.nlm.nih.gov/articles/PMC11034792/
  55. https://www.embopress.org/doi/10.1038/embor.2009.90
  56. https://www.cell.com/molecular-cell/fulltext/S1097-2765(17)30173-9
  57. https://www.nature.com/articles/s41467-023-39846-y
  58. https://www.nature.com/articles/s41467-020-15006-4
  59. https://www.cell.com/fulltext/S0092-8674%2800%2981656-6
  60. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262277
  61. https://pmc.ncbi.nlm.nih.gov/articles/PMC8903295/
  62. https://epigeneticsandchromatin.biomedcentral.com/articles/10.1186/1756-8935-6-32
  63. https://www.nature.com/articles/s41586-023-06872-1
  64. https://genesdev.cshlp.org/content/17/3/330.short
  65. https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2025.1548946/full
  66. https://www.sciencedirect.com/science/article/pii/S2211124723000074
  67. https://www.pnas.org/doi/10.1073/pnas.2211855120
  68. https://www.embopress.org/doi/full/10.1038/s44319-024-00085-x
  69. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02106-z
  70. https://doi.org/10.1126/science.1083430
  71. https://doi.org/10.1101/gad.1547007
  72. https://pmc.ncbi.nlm.nih.gov/articles/PMC5796526/
  73. https://doi.org/10.1101/gad.477208
  74. https://pmc.ncbi.nlm.nih.gov/articles/PMC5333063/
  75. https://doi.org/10.1038/nrm.2017.67
  76. https://doi.org/10.1128/MCB.21.13.4129-4139.2001
  77. https://academic.oup.com/nar/article/40/2/477/2409009
  78. https://doi.org/10.1101/gad.1666208
  79. https://elifesciences.org/articles/04766
Add your contribution
Related Hubs
User Avatar
No comments yet.