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Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)—the epigenetic determinant of centromere identity—is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of ‘mitotic drive’ that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs., Author summary Stem cells can divide in an asymmetric fashion giving rise to two daughter cells with different fates. One daughter remains a stem cell, while the other can differentiate and adopt a new cell fate. Germline stem cells in the testes and ovaries give rise to differentiating daughter cells that eventually form the gametes, eggs and sperm. Here we investigate mechanisms controlling germline stem cell divisions occurring in the ovary of the fruit fly Drosophila melanogaster. Centromeres are epigenetically specified loci on chromosomes that make essential connections to the cell division machinery. Our study is focused on the centromere component CENP-C. We show that CENP-C is critical for the correct assembly of centromeres that occurs prior to cell division in germline stem cells. In addition, we find that CENP-C is asymmetrically distributed between stem and daughter cells, with more CENP-C at stem cell centromeres. Finally, we show that CENP-C depletion in germline stem cells disrupts the balance of stem and daughter cells in the developing ovary, impacting on cell fate. Taken together, we propose that CENP-C level and function at centromeres plays an important role in determining cell fate upon asymmetric division occurring in stem cells.

Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins’ ability to recruit ORC and compete for MCM when MCM becomes limiting., Author summary The coordinated replication of complex eukaryotic genomes is primarily regulated at the level of replication initiation. Therefore, how initiation is regulated in time and space is an active field of research. A leading hypothesis is that the stoichiometry of MCM, the replicative helicase, at replication origins is a significant determinant of initiation timing. However, how MCM stoichiometry at origins is regulated is unknown. We tested two hypothetical mechanisms for the control of MCM stoichiometry: Origin capacity, in which MCM stoichiometry is constrained by the varying capacity of individual origins to load MCM, and ORC activity, in which MCM stoichiometry is regulated by the varying activity of ORC, the MCM loader, at individual origins. We find that neither mechanism is consistent with our data, but that a hybrid of the two mechanisms is.

During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader., Author summary Within the cell’s nucleus the DNA is wrapped around proteins called histones. Upon DNA replication, newly synthesized H3 histones are acetylated at lysine 56. This acetylation is significant for the cell because when it is not removed in a timely manner it leads to genomic instability. We have investigated the source of this instability and discovered that the kinase Dun1, usually implicated in the regulation of dNTPs, the building blocks of DNA, has a novel, dNTP-independent, essential role when histones are hyper-acetylated. The essential role of Dun1 is in the regulation of the temporal program of DNA replication. Thus, our results uncover what the main defect is in cells unable to regulate the acetylation of histones, while revealing new functions for well-characterized proteins with roles in genome stability maintenance.

DNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability., Author summary DNA replication in eukaryotes is carried out by the replisome–a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol δ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol δ cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol δ in budding yeast–a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol δ scarcity. The defects we observe provide insight into how destabilizing Pol δ mutations lead to genome instability.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes., Author summary Gene-rich regions of the genome tend to replicate earlier in S phase than do repetitive and other non-genic regions. This may be an evolutionary consequence of the fact that replication later in S phase is associated with higher frequencies of mutation and genome rearrangement. Replication timing along the chromosome is determined by 1) events prior to S-phase that specify the locations where DNA replication can be initiated, referred to as origin licensing; and 2) the timing of activation of these licensed origins during S-phase, referred to as origin firing. To determine the relative importance of these two mechanisms, here we identify both the binding sites and the abundance of a key component of the origin licensing machinery in budding yeast, fission yeast, mice, and humans, namely the replicative helicase complex. We discovered that, with a few notable exceptions, which include the inactive X chromosome in mammals, the program of replication timing can be largely explained simply on the basis of origin licensing. Our results support a model for replication timing that emphasizes stochastic firing of origins that have been licensed before S phase begins.

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting., Author summary Half of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle., Author summary Structure-specific endonucleases are enzymes that process DNA intermediates generated in DNA replication, recombination, and repair. Proper regulation of these enzymes is critical for maintaining genome stability. Dna2 and XPF are two such enzymes present across eukaryotes, from yeasts to humans. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of Dna2 and Rad16 (the equivalent of human XPF) are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin E3 ligase. In the S phase of the cell cycle, CRL4Cdt2 promotes the degradation of Pxd1, which is an inhibitor of Dna2 and an activator of Rad16. Through targeting Pxd1 for degradation, CRL4Cdt2 increases the activity of Dna2 in S phase and is important for the normal S-phase function of Dna2. Meanwhile, the degradation of Pxd1 reduces the activity of Rad16 in S phase, and curtails Rad16-dependent single-strand annealing, a mutagenic DNA repair pathway. Our findings uncover a new mechanism regulating two important endonucleases during the cell cycle, and reveal a new way of coordinating endonucleases to safeguard genome stability.

DNA polymerase epsilon (Pol ε) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ε catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability., Author summary DNA replication factors are tightly regulated to ensure genome duplication accuracy and efficiency. Among these factors, the Pol ε replicative polymerase plays a vital role by copying half of the genome every cell cycle. However, little is known about how this critical enzyme is regulated. Here we describe SUMO-based regulation of the catalytic subunit of Pol ε, Pol2. Our data suggest that Pol2 sumoylation occurs during replication elongation, particularly when replication forks encounter template obstacles. This modification is mediated by the conserved Smc5/6 SUMO ligase complex and occurs at a single site within the Pol2 catalytic domain. Several observations suggest that Pol2 sumoylation makes positive contributions to the synthesis of DNA regions enriched with template barriers and helps to prevent large-scale genomic alterations. Our work thus provides new insights into DNA polymerase regulation, specifically the role played by contributions from SUMO and the Smc5/6 complex.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors., Author summary In order to survive exposure to environmental stress, cells transiently arrest the cell division cycle while they adapt to the stress. Several kinases and phosphatases are known to control stress adaptation programs, but the extent to which these signaling pathways work together to tune the stress response is not well understood. This study investigates the role of the phosphatase calcineurin in the stress response and shows that calcineurin inhibits the cell cycle in part by stimulating the activity of the Hog1/p-38 stress-activated MAPK in both yeast and human cells. Crosstalk between stress response pathways may help cells mount specific responses to diverse stressors and to survive changes in their environment.

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans., Author summary The integrity of DNA is essential for life so even the most primitive life forms have evolved a DNA repair ‘toolkit’ to detect and fix different types of DNA damage. XPF-ERCC1 is an enzyme that can cut DNA and a key player in many of these DNA repair transactions. Consistent with this, inactivating mutations of XPF-ERCC1 in humans and mice lead to a dramatic premature ageing phenotype associated with liver, kidney and bone marrow dysfunction. Here, we ask which of the many functions of XPF-ERCC1 are required to protect tissues from endogenous DNA damage. To do this, we generated cells and mice lacking two of the best characterised functions of XPF-ERCC1: nucleotide excision repair and inter-strand crosslink repair. Surprisingly, neither the cells nor mice lacking these two repair pathways behave like the XPF-ERCC1 mutants, in fact the mice are remarkable in their lack of phenotype. Our work suggests that there are functions of XPF-ERCC1 outside of the canonical repair pathways which are important for DNA repair and the homeostasis of multiple organs.

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome., Author summary Eukaryotic chromosome duplication begins at sites called origins of replication along the chromosomal DNA. A conserved property of eukaryotic origins is that they vary in efficiency—the proportion of cells in a population in which they “fire”—and in the average time of activation within S phase, but the molecular details underlying this variation are not well understood. Previous work has shown that limiting concentrations of a set of conserved replication initiation proteins referred to as “SSDDCS” (Sld2, Sld3, Dbf4, Dpb11, Cdc45, and Sld7) are rate limiting for origin activation in budding yeast and other eukaryotes; combined overexpression of these proteins increases and/or advances origin firing. However, it remained possible that different factors affect different aspects of origin activation (e.g., timing vs. efficiency). Here, by depleting individual factors or by overexpressing sets of factors in budding yeast, we demonstrate that it is levels of Sld3, Cdc45 and/or Sld7 levels are primarily responsible for modulating the differences in relative origin efficiency and timing. This work gives further insights into what shapes the landscape of genome duplication.

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis., Author summary The appropriate amount and balance of four dNTPs are crucial for all cells correctly copying and passing on their genetic material generation by generation. Eukaryotes have developed an alert and response system to deal with the disturbance. Here, we uncovered a second-level effector branch. It is activated by the upstream surveillance kinase cascade, which can induce the expression of dNTP-producing enzymes. It can also reduce the inhibitor of these enzymes to further boost their activity according to the degrees of threats. These findings suggest a multi-level response system to guarantee the appropriate dNTP supply, which is essential to maintain genetic stability under various environmental challenges.

Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity., Author summary The migratory locust, Locusta migratoria, is a worldwide agricultural pest that displays a remarkable density-dependent phase polyphenism, where being kept in a crowd triggers individuals to transit from the sedentary solitarious phase into the high-active gregarious phase. So, the migratory locust has been regarded as an excellent study model for environment-induced behavioral plasticity. Our previous finding shows that NPF2-regulated NOS transcription plays important roles in phase-related locomotor plasticity in the locust. Here, we further demonstrate that phosphorylated CREB-B directly activates NOS transcription in the pars intercerebralis, thus mediates phase-related locomotor plasticity. Further studies show that the levels of CREB-B phosphorylation is positively correlated with the crowding treatment and suppressed by NPF2. Among several candidate kinases, protein kinase A is demonstrated to transmit the inhibitory effects of NPF2 on CREB-B phosphorylation and NOS transcription. Our study provides deep insight into the precise regulatory mechanisms underlying environment-induced behavioral plasticity.

The comorbid association of autoimmune diseases with cancers has been a major obstacle to successful anti-cancer treatment. Cancer survival rate decreases significantly in patients with preexisting autoimmunity. However, to date, the molecular and cellular profiles of such comorbidities are poorly understood. We used Aicardi-Goutières syndrome (AGS) as a model autoimmune disease and explored the underlying mechanisms of genome instability in AGS-associated-gene-deficient patient cells. We found that R-loops are highly enriched at transcription-replication conflict regions of the genome in fibroblast of patients bearing SAMHD1 mutation, which is the AGS-associated-gene mutation most frequently reported with tumor and malignancies. In SAMHD1-depleted cells, R-loops accumulated with the concomitant activation of DNA damage responses. Removal of R-loops in SAMHD1 deficiency reduced cellular responses to genome instability. Furthermore, downregulation of SAMHD1 expression is associated with various types of cancer and poor survival rate. Our findings suggest that SAMHD1 functions as a tumor suppressor by resolving R-loops, and thus, SAMHD1 and R-loop may be novel diagnostic markers and targets for patient stratification in anti-cancer therapy., Author summary Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), a monogenic lupus-like autoimmune disease. Among AGS-associated genes, SAMHD1 is most frequently mutates in various types of tumors and malignancies, suggesting that it is biologically relevant to cancer development. Here, we show that SAMHD1 resolves R-loops induced by transcription-replication conflicts, thereby contributing to the maintenance of genome stability. Our findings provide insight into the molecular and mechanical understanding of the autoimmunity and cancer comorbidity, and suggest that SAMHD1 and R-loops are potential and reliable biomarkers in anti-cancer therapeutics.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication., Author summary The initial step of DNA replication is loading the DNA helicase, MCM, onto DNA during the first phase of the cell division cycle. If MCM loading occurs inappropriately onto DNA that has already been replicated, then cells risk DNA re-replication, a source of endogenous DNA damage and genome instability. How mammalian cells prevent any sections of their very large genomes from re-replicating is still not fully understood. We found that the Cdt1 protein, one of the critical MCM loading factors, is inhibited specifically in late cell cycle stages through a mechanism involving protein phosphorylation. This phosphorylation prevents Cdt1 from binding MCM; when Cdt1 cannot be phosphorylated MCM is inappropriately re-loaded onto DNA and cells are prone to re-replication. When cells divide and transition into G1 phase, Cdt1 is then dephosphorylated to re-activate it for MCM loading. Based on these findings we assert that the different mechanisms that cooperate to avoid re-replication are not redundant. Instead, different cell cycle phases are dominated by different re-replication control mechanisms. These findings have implications for understanding how genomes are duplicated precisely once per cell cycle and shed light on how that process is perturbed by changes in Cdt1 levels or phosphorylation activity.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role., Author summary DNA replication is the central process that duplicates the genetic information during cell multiplication. Many cellular factors play important roles in the efficient and accurate duplication of DNA, critical for faithful transmission of genetic information. One such factor is Elg1. Elg1 acts to unload PCNA, the ring-shaped processivity factor that holds DNA polymerases on DNA for replication. In this work, we identify an additional role for Elg1 during replication. We show that lack of Elg1 leads to defects in packaging of DNA into chromatin after DNA replication. In addition, we found that Elg1 interacts with histone chaperones, factors which play key role in chromatin formation. Examining causes of the chromatin re-assembly defect, we show that accumulation of PCNA on DNA is the main cause of defective chromatin formation in the absence of Elg1. By uncovering a new route through which Elg1 ensures chromosomes are perfectly copied, our findings advance understanding of how Elg1 contributes to the stability of the genome through its key roles in DNA replication.

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., Author summary Proliferating cells duplicate their genetic material via a highly-ordered process called DNA replication. Genetic lesions caused by a variety of environmental chemicals can inhibit DNA replication progression, thereby causing genetic abnormalities and cell death, as well as promoting the development of diseases such as cancer. To fit within the confines of the cell’s nucleus, DNA is wrapped around proteins called histones, which play critical roles in promoting accurate DNA replication. In this study, we reveal unexpected functional links between histones and cellular factors that regulate telomeres, which are structures protecting the extremities of DNA molecules in cells. We demonstrate that a functional interplay between histone- and telomere-regulating factors allow cells to duplicate their genetic material in a timely manner in the presence of replication-blocking chemicals. Overall, our study highlights new mechanisms through which proliferating cells avoid the deleterious consequences associated with compromised DNA replication.

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS., Author summary Chromosomes are DNA molecules that contain the genetic information. During replication, the two sister DNA molecules covered by proteins (sister chromatids) are held together by many copies of a ring-like protein complex named cohesin, in a process called sister-chromatid cohesion. Before a cell divides, the cohesin rings are removed from the two sister chromatids to allow their migration towards the opposite poles of the dividing mother cell. At the end of this process, the two daughter cells have inherited a complete set of chromosomes. Before the next cell division, chromosomes are duplicated with high speed and fidelity. This important task is performed by the DNA replication machinery, a sophisticated apparatus made of several enzymes and proteins. In the present study, we have demonstrated that DDX11 and Timeless, two subunits of the DNA replication machinery, recruit the cohesin rings to promote their stable binding to the newly duplicated chromosomes, that is the establishment of sister-chromatid cohesion. In human cells that were genetically engineered to reduce the level of DDX11, we observed that sister-chromatid cohesion was loosened and association of cohesin to chromosomes was reduced. Our experimental results contribute to our understanding of the molecular mechanisms underlying the functional coupling between DNA replication and sister-chromatid cohesion in human cells.

Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth., Author summary Smc5/6 belongs to the SMC (Structural Maintenance of Chromosomes) family of protein complexes, all of which are highly conserved and critical for genome maintenance. To address the roles of Smc5/6 during growth, we rapidly depleted its subunits in yeast and found the main acute effect to be defective ribosomal DNA (rDNA) duplication. The rDNA contains hundreds of sites that can pause replication forks; these must be carefully managed for cells to finish replication. We found that reducing fork pausing improved rDNA replication in cells without Smc5/6. Further analysis suggested that Smc5/6 prevents the DNA helicase Mph1 from turning paused forks into recombination structures, which cannot be processed without Smc5/6. Our findings thus revealed a key role for Smc5/6 in managing endogenous replication fork pausing. As rDNA and its associated nucleolar structure are critical for overall genome maintenance and other cellular processes, rDNA regulation by Smc5/6 would be expected to have multilayered effects on cell physiology and growth.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis., Author summary The origin recognition complex (ORC) is essential for licensing replication origins during M/G1 for their firing in the subsequent S phase. Individuals with a rare form of dwarfism called Meier-Gorlin syndrome (MGS) have mutations in proteins required for origin activation, including various subunits of ORC. To better understand the molecular and cellular consequences of these mutations, we introduced an equivalent MGS mutation in ORC4 into yeast. We find that origin activity in the ribosomal DNA (rDNA) repeats is severely compromised in yeast that harbor the MGS allele. Consequently, cells that have reduced their rDNA copy number from ~150 to fewer than 30 copies overtake the culture. Although the loss of rDNA repeats helps ensure the complete replication of chromosome XII during S phase, cells with fewer rDNA repeats struggle to meet the high demand for ribosomal RNA.