Replication Origin Firing Research Articles

Arabidopsis DNA Replication Initiates in Intergenic, AT-Rich Open Chromatin.

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

Abstract PL03-02: Exploiting DNA replication stress for cancer therapies

Abstract A feature of most cancers is genomic instability. We and others have proposed a model to explain the presence of such genomic instability. Briefly, we proposed that oncogenes, which are activated in practically all cancers and drive cell proliferation, induce DNA replication stress. The presence of DNA replication stress leads to the formation of DNA double-strand breaks, which, when incorrectly repaired, lead to genomic instability. DNA replication stress is a somewhat non-specific term used to indicate that cells have difficulties to replicate their DNA. Inhibitors of DNA polymerases slow down DNA replication and induce DNA replication stress. However, in this case, the replisome, i.e. the proteins that replicate the DNA, remains intact and DNA double-strand breaks are not formed. Oncogenes induce a different type of DNA replication stress, as select replisomes are disrupted, leaving the DNA “naked” and susceptible to DNA double-strand break formation. Recent results from a number of laboratories have provided great insights on how oncogenes induce DNA replication stress. Specifically, it has been found that oncogenes induce the firing of novel DNA replication origins. These oncogene-induced origins are present within transcribed genes. When these origins fire and DNA replication begins, the replisomes collide with the transcription machinery. These aberrant collisions lead to disruption of the replisome. Disrupted, aka as collapsed, replisomes need to be repaired for DNA replication to restart. Errors in this repair lead to DNA double-strand breaks and genomic instability. The main pathway for repair of collapsed DNA replication forks appears to be break-induced replication. This pathway is used very infrequently by normal cells and, as such, certain proteins that function in this pathway are not essential for normal development. However, their inactivation suppresses cancer development, because cancer cells rely on break-induced replication to repair their collapsed DNA replication forks. It follows that proteins that function in break-induced replication may be good targets for the development of effective, non-toxic cancer therapies. Citation Format: Thanos Halazonetis. Exploiting DNA replication stress for cancer therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL03-02. doi:10.1158/1535-7163.TARG-19-PL03-02

Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery.

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

Abstract LB-288: CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics

Abstract DNA replication is a tightly regulated process required for faithful duplication of the genome. Previous research in lower eukaryotes has demonstrated a requisite role for the CDC7 protein kinase in DNA replication origin firing through phosphorylation of the MCM2-7 helicase complex. Owing to its overexpression in various neoplasms, CDC7 has emerged as an attractive target for cancer treatment. We previously reported that SRA141, a potent and selective CDC7 inhibitor, is cytotoxic to multiple tumor cell lines in vitro and demonstrates robust anti-tumor efficacy in colorectal and leukemia xenograft models. Here, we explored the effects of SRA141 on DNA replication and cell cycle dynamics and how these effects relate to the mechanism of SRA141-induced cell death in colorectal cancer cell lines. SRA141 treatment caused a complete inhibition of MCM2 phosphorylation and reduced overall cellular DNA synthesis within 3 hours, with the greatest effects observed in late S-phase. Unexpectedly, DNA combing experiments indicated that SRA141 caused a 70-90% increase in the rate of replication fork progression. These findings suggest that SRA141 may indeed limit origin firing, but this effect is compensated for in part by increased replication fork rates. Despite delay, nucleotide analog pulse-chase experiments indicated that SRA141 treated cells ultimately finished S phase and transitioned into mitosis. In contrast to other reports, no evidence of replication fork collapse was observed, indicating that the main mechanism of SRA141 cytotoxicity is not mediated through replication fork collapse. Intriguingly, some of the most pronounced effects of SRA141 treatment were on progression through M phase, as evidenced by an accumulation of mitotic cells and increased Cyclin B levels following treatment. Moreover, inhibition of Aurora B kinase, which regulates mitotic progression, strongly synergized with SRA141 in caspase-dependent cancer cell killing. Taken together, our findings indicate that the mechanism of cytotoxicity of CDC7 inhibitors is distinct from agents that cause replication fork collapse or inhibit CDKs, and thus may define a new class of cancer therapeutic agents that synergize with drugs that target certain mitotic pathways. Citation Format: Veena Jagannathan, Snezana Milutinovic, Ryan Hansen, Bryan Strouse, Christian Hassig, Eric Brown. CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-288.

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2–7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2–7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

An essential role for dNTP homeostasis following CDK-induced replication stress.

ABSTRACTReplication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a ‘dNTP supply and demand’ model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

Origin Firing Regulations to Control Genome Replication Timing.

Complete genome duplication is essential for genetic homeostasis over successive cell generations. Higher eukaryotes possess a complex genome replication program that involves replicating the genome in units of individual chromatin domains with a reproducible order or timing. Two types of replication origin firing regulations ensure complete and well-timed domain-wise genome replication: (1) the timing of origin firing within a domain must be determined and (2) enough origins must fire with appropriate positioning in a short time window to avoid inter-origin gaps too large to be fully copied. Fundamental principles of eukaryotic origin firing are known. We here discuss advances in understanding the regulation of origin firing to control firing time. Work with yeasts suggests that eukaryotes utilise distinct molecular pathways to determine firing time of distinct sets of origins, depending on the specific requirements of the genomic regions to be replicated. Although the exact nature of the timing control processes varies between eukaryotes, conserved aspects exist: (1) the first step of origin firing, pre-initiation complex (pre-IC formation), is the regulated step, (2) many regulation pathways control the firing kinase Dbf4-dependent kinase, (3) Rif1 is a conserved mediator of late origin firing and (4) competition between origins for limiting firing factors contributes to firing timing. Characterization of the molecular timing control pathways will enable us to manipulate them to address the biological role of replication timing, for example, in cell differentiation and genome instability.

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Endoreduplication of the mouse genome in the absence of ORC1.

The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.

Claspin - checkpoint adaptor and DNA replication factor.

Claspin was discovered as a Chk1-interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single-stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint-mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

The time-dependent rate [Formula: see text] of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here, we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the [Formula: see text] universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the [Formula: see text] maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis

SummaryDNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4−/− mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4−/−p53+/− mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

An interplay between multiple sirtuins promotes completion of DNA replication in cells with short telomeres.

The evolutionarily-conserved sirtuin family of histone deacetylases regulates a multitude of DNA-associated processes. A recent genome-wide screen conducted in the yeast Saccharomyces cerevisiae identified Yku70/80, which regulate nonhomologous end-joining (NHEJ) and telomere structure, as being essential for cell proliferation in the presence of the pan-sirtuin inhibitor nicotinamide (NAM). Here, we show that sirtuin-dependent deacetylation of both histone H3 lysine 56 and H4 lysine 16 promotes growth of yku70Δ and yku80Δ cells, and that the NAM sensitivity of these mutants is not caused by defects in DNA double-strand break repair by NHEJ, but rather by their inability to maintain normal telomere length. Indeed, our results indicate that in the absence of sirtuin activity, cells with abnormally short telomeres, e.g., yku70/80Δ or est1/2Δ mutants, present striking defects in S phase progression. Our data further suggest that early firing of replication origins at short telomeres compromises the cellular response to NAM- and genotoxin-induced replicative stress. Finally, we show that reducing H4K16ac in yku70Δ cells limits activation of the DNA damage checkpoint kinase Rad53 in response to replicative stress, which promotes usage of translesion synthesis and S phase progression. Our results reveal a novel interplay between sirtuin-mediated regulation of chromatin structure and telomere-regulating factors in promoting timely completion of S phase upon replicative stress.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

DNA Replication: Onco-agent provocateur.

Oncogenes induce firing of replication origins at transcribed genes; this promotes transcription–replication conflicts and genome instability.

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2—but not FANCI—plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Firing of Replication Origins Frees Dbf4-Cdc7 to Target Eco1 for Destruction

Robust progression through the cell-division cycle depends on the precisely ordered phosphorylation of hundreds of different proteins by cyclin-dependent kinases (CDKs) and other kinases. The order of CDK substrate phosphorylation depends on rising CDK activity, coupled with variations in substrate affinities for different CDK-cyclin complexes and the opposing phosphatases [1-4]. Here, we address the ordering of substrate phosphorylation by a second major cell-cycle kinase, Cdc7-Dbf4 or Dbf4-dependent kinase (DDK). The primary function of DDK is to initiate DNA replication by phosphorylating the Mcm2-7 replicative helicase [5-7]. DDK also phosphorylates the cohesin acetyltransferase Eco1 [8]. Sequential phosphorylations of Eco1 by CDK, DDK, and Mck1 create a phosphodegron that isrecognized by the ubiquitin ligase SCFCdc4. DDK, despite being activated in early S phase, does not phosphorylate Eco1 to trigger its degradation until late S phase [8]. DDK associates with docking sites on loaded Mcm double hexamers at unfired replication origins [9, 10]. We hypothesized that these docking interactions sequester limiting amounts of DDK, delaying Eco1 phosphorylation by DDK until replication is complete. Consistent with this hypothesis, we find that overproduction of DDK leads to premature Eco1 degradation. Eco1 degradation also occurs prematurely if Mcm complex loading at origins is prevented by depletion of Cdc6, and Eco1 is stabilized if loaded Mcm complexes are prevented from firing by a Cdc45 mutant. We propose that the timing of Eco1 phosphorylation, and potentially that of other DDK substrates, is determined in part by sequestration of DDK at unfired replication origins during S phase.

Cellular Detection of G-Quadruplexes by Optical Imaging Methods.

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription

SummaryChromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.