Revised mechanism of hydroxyurea-induced cell cycle arrest and an improved alternative.

Replication stress describes endogenous and exogenous challenges to DNA replication in the S-phase. Stress during this critical process causes helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response in yeast is hydroxyurea (HU), a clinically used chemotherapeutic. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of deoxynucleotide triphosphate synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, facilitated by the replisome-associated mediator Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast and human cells and show that antioxidants restore growth of yeast in HU. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol α, Pol δ, and Pol ε, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron-sulfur (Fe-S) clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.

Inferring replication timing and proliferation dynamics from single-cell DNA sequencing data.

Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in  >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors.

The G1-S transition is promoted by Rb degradation via the E3 ligase UBR5.

Mammalian cells make the decision to divide at the G1-S transition in response to diverse signals impinging on the retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Passage through the G1-S transition is initially driven by Rb inactivation via phosphorylation and by Rb's decreasing concentration in G1. While many studies have identified the mechanisms of Rb phosphorylation, the mechanism underlying Rb's decreasing concentration in G1 was unknown. Here, we found that Rb's concentration decrease in G1 requires the E3 ubiquitin ligase UBR5. UBR5 knockout cells have increased Rb concentration in early G1, exhibited a lower G1-S transition rate, and are more sensitive to inhibition of cyclin-dependent kinase 4/6 (Cdk4/6). This last observation suggests that UBR5 inhibition can strengthen the efficacy of Cdk4/6 inhibitor-based cancer therapies.

Aurora A Kinase Begins to Localize to the Centrosome in the S-phase of the Cell Cycle in the XL2 Cell Line.

BACKGROUND: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. METHODS: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. RESULTS: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. CONCLUSIONS: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.

Replication fork stalling in late S-phase elicits nascent strand degradation by DNA mismatch repair.

Eukaryotic chromosomal replication occurs in a segmented, temporal manner wherein open euchromatin and compact heterochromatin replicate during early and late S-phase respectively. Using single molecule DNA fiber analyses coupled with cell synchronization, we find that newly synthesized strands remain stable at perturbed forks in early S-phase. Unexpectedly, stalled forks are susceptible to nucleolytic digestion during late replication resulting in defective fork restart. This inherent vulnerability to nascent strand degradation is dependent on fork reversal enzymes and resection nucleases MRE11, DNA2 and EXO1. Inducing chromatin compaction elicits digestion of nascent DNA in response to fork stalling due to reduced association of RAD51 with nascent DNA. Furthermore, RAD51 occupancy at stalled forks in late S-phase is diminished indicating that densely packed chromatin limits RAD51 accessibility to mediate replication fork protection. Genetic analyses reveal that susceptibility of late replicating forks to nascent DNA digestion is dependent on EXO1 via DNA mismatch repair (MMR) and that the BRCA2-mediated replication fork protection blocks MMR from degrading nascent DNA. Overall, our findings illustrate differential regulation of fork protection between early and late replication and demonstrate nascent strand degradation as a critical determinant of heterochromatin instability in response to replication stress.

Embryonic genome instability upon DNA replication timing program emergence.

Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Embryonic stem cells maintain high origin activity and slow forks to coordinate replication with cell cycle progression.

Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication.

APC/C prevents a noncanonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest.

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.

DNA polymerase κ participates in early S-phase DNA replication in human cells.

Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N 2-heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N 2-modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

p53 ensures the normal behavior and modification of G1/S-specific histone H3.1 in the nucleus.

H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.

Quantifying DNA replication speeds in single cells by scEdU-seq.

In a human cell, thousands of replication forks simultaneously coordinate duplication of the entire genome. The rate at which this process occurs might depend on the epigenetic state of the genome and vary between, or even within, cell types. To accurately measure DNA replication speeds, we developed single-cell 5-ethynyl-2'-deoxyuridine sequencing to detect nascent replicated DNA. We observed that the DNA replication speed is not constant but increases during S phase of the cell cycle. Using genetic and pharmacological perturbations we were able to alter this acceleration of replication and conclude that DNA damage inflicted by the process of transcription limits the speed of replication during early S phase. In late S phase, during which less-transcribed regions replicate, replication accelerates and approaches its maximum speed.

Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

Inhibition of the ATR-DNAPKcs-RB axis drives G1/S-phase transition and sensitizes triple-negative breast cancer (TNBC) to DNA holliday junctions.

Targeting the DNA damage response (DDR) is a promising strategy in oncotherapy, as most tumor cells are sensitive to excess damage due to their repair defects. Ataxia telangiectasia mutated and RAD3-related protein (ATR) is a damage response signal transduction sensor, and its therapeutic potential in tumor cells needs to be precisely investigated. Herein, we identified a new axis that could be targeted by ATR inhibitors to decrease the DNA-dependent protein kinase catalytic subunit (DNAPKcs), downregulate the expression of the retinoblastoma (RB), and drive G1/S-phase transition. Four-way DNA Holliday junctions (FJs) assembled in this process could trigger S-phase arrest and induce lethal chromosome damage in RB-positive triple-negative breast cancer (TNBC) cells. Furthermore, these unrepaired junctions also exerted toxic effects to RB-deficient TNBC cells when the homologous recombination repair (HRR) was inhibited. This study proposes a precise strategy for treating TNBC by targeting the DDR and extends our understanding of ATR and HJ in tumor treatment.

Triple lysine and nucleosome-binding motifs of the viral IE19 protein are required for human cytomegalovirus S-phase infections.

Herpesvirus genomes are maintained as extrachromosomal plasmids within the nuclei of infected cells. Some herpesviruses persist within dividing cells, putting the viral genome at risk of being lost to the cytoplasm during mitosis because karyokinesis (nuclear division) requires nuclear envelope breakdown. Oncogenic herpesviruses (and papillomaviruses) avoid genome loss during mitosis by tethering their genomes to cellular chromosomes, thereby ensuring viral genome uptake into newly formed nuclei. These viruses use viral proteins with DNA- and chromatin-binding capabilities to physically link viral and cellular genomes together in a process called tethering. The known viral tethering proteins of human papillomavirus (E2), Epstein-Barr virus (EBNA1), and Kaposi's sarcoma-associated herpesvirus (LANA) each contain two independent domains required for genome tethering, one that binds sequence specifically to the viral genome and another that binds to cellular chromatin. This latter domain is called a chromatin tethering domain (CTD). The human cytomegalovirus UL123 gene encodes a CTD that is required for the virus to productively infect dividing fibroblast cells within the S phase of the cell cycle, presumably by tethering the viral genome to cellular chromosomes during mitosis. The CTD-containing UL123 gene product that supports S-phase infections is the IE19 protein. Here, we define two motifs in IE19 required for S-phase infections: an N-terminal triple lysine motif and a C-terminal nucleosome-binding motif within the CTD.IMPORTANCEThe IE19 protein encoded by human cytomegalovirus (HCMV) is required for S-phase infection of dividing cells, likely because it tethers the viral genome to cellular chromosomes, thereby allowing them to survive mitosis. The mechanism through which IE19 tethers viral genomes to cellular chromosomes is not understood. For human papillomavirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, viral genome tethering is required for persistence (latency) and pathogenesis (oncogenesis). Like these viruses, HCMV also achieves latency, and it modulates the properties of glioblastoma multiforme tumors. Therefore, defining the mechanism through which IE19 tethers viral genomes to cellular chromosomes may help us understand, and ultimately combat or control, HCMV latency and oncomodulation.

APC/C-regulated CPT1C promotes tumor progression by upregulating the energy supply and accelerating the G1/S transition.

BACKGROUND: In addition to functioning as a precise monitoring mechanism in cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is reported to be involved in regulating multiple metabolic processes by facilitating the ubiquitin-mediated degradation of key enzymes. Fatty acid oxidation is a metabolic pathway utilized by tumor cells that is crucial for malignant progression; however, its association with APC/C remains to be explored. METHODS: Cell cycle synchronization, immunoblotting, and propidium iodide staining were performed to investigate the carnitine palmitoyltransferase 1 C (CPT1C) expression manner. Proximity ligation assay and co-immunoprecipitation were performed to detect interactions between CPT1C and APC/C. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assays, cell-scratch assays, and transwell assays and xenograft transplantation assays were performed to investigate the role of CPT1C in tumor progression in vitro and in vivo. Immunohistochemistry was performed on tumor tissue microarray to evaluate the expression levels of CPT1C and explore its potential clinical value. RESULTS: We identified CPT1C as a novel APC/C substrate. CPT1C protein levels exhibited cell cycle-dependent fluctuations, peaking at the G1/S boundary. Elevated CPT1C accelerated the G1/S transition, facilitating tumor cell proliferation in vitro and in vivo. Furthermore, CPT1C enhanced fatty acid utilization, upregulated ATP levels, and decreased reactive oxygen species levels, thereby favoring cell survival in a harsh metabolic environment. Clinically, high CPT1C expression correlated with poor survival in patients with esophageal squamous cell carcinoma. CONCLUSIONS: Overall, our results revealed a novel interplay between fatty acid utilization and cell cycle machinery in tumor cells. Additionally, CPT1C promoted tumor cell proliferation and survival by augmenting cellular ATP levels and preserving redox homeostasis, particularly under metabolic stress. Therefore, CPT1C could be an independent prognostic indicator in esophageal squamous cell carcinoma.

A reverse brake for the cell cycle.

Mitogenic signaling acts beyond S-phase entry to prevent whole-genome duplications.

CDK4/6 activity is required during G2 arrest to prevent stress-induced endoreplication.

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling.

Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression.

BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.