Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Delta cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Delta cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Delta cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Delta cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.     

The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1)

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.     

Dormant origin signaling during unperturbed replication

Mechanisms that limit origin firing are essential as the ˜50,000 origins that replicate the human genome in unperturbed cells are chosen from an excess of ˜500,000 licensed origins. Computational models of the spatiotemporal pattern of replication foci assume that origins fire stochastically with a domino-like progression that places later firing origins near recent fired origins. These stochastic models of origin firing require dormant origin signaling that inhibits origin firing and suppresses licensed origins for passive replication at a distance of ∼7–120 kbp around replication forks. ATR and CHK1 kinase inhibitors increase origin firing and increase origin density in unperturbed cells. Thus, basal ATR and CHK1 kinase-dependent dormant origin signaling inhibits origin firing and there appear to be two thresholds of ATR kinase signaling. A minority of ATR molecules are activated for ATR and CHK1 kinase-dependent dormant origin signaling and this is essential for DNA replication in unperturbed cells. A majority of ATR molecules are activated for ATR and CHK1 kinase-dependent checkpoint signaling in cells treated with DNA damaging agents that target replication forks. Since ATR and CHK1 kinase inhibitors increase origin firing and this is associated with fork stalling and extensive regions of single-stranded DNA, they are DNA damaging agents. Accordingly, the sequence of administration of ATR and CHK1 kinase inhibitors and DNA damaging agents may impact the DNA damage induced by the combination and the efficacy of cell killing by the combination.     

Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing

Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.     

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ～10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.     

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.     

Open sesame: activating dormant replication origins in the mouse immunoglobulin heavy chain (Igh) locus

Chromosomal DNA replication in mammals initiates from replication origins whose activity differs in accordance with cell type and differentiation state. In addition to origins that are active in unperturbed conditions, chromosomes also contain dormant origins that can become functional in response to certain genotoxic stress conditions. Improper regulation of origin usage can cause genomic instability leading to tumorigenesis. We review findings from recent single-molecule DNA fiber studies examining replication of the mouse immunoglobulin heavy chain (Igh) locus, in which origin activity over a 400kb region is subject to dramatic developmental regulation. Possible models are discussed to explain such differential origin usage, particularly during replication stress conditions that can activate dormant origins.     

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.     

The Hsk1(Cdc7) replication kinase regulates origin efficiency

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.     

Genome-wide estimation of firing efficiencies of origins of DNA replication from time-course copy number variation data

Background  

DNA replication is a fundamental biological process during S phase of cell division. It is initiated from several hundreds of origins along whole chromosome with different firing efficiencies (or frequency of usage). Direct measurement of origin firing efficiency by techniques such as DNA combing are time-consuming and lack the ability to measure all origins. Recent genome-wide study of DNA replication approximated origin firing efficiency by indirectly measuring other quantities related to replication. However, these approximation methods do not reflect properties of origin firing and may lead to inappropriate estimations.     

Human CST abundance determines recovery from diverse forms of DNA damage and replication stress

Mammalian CST (CTC1-STN1-TEN1) is a telomere-associated complex that functions in telomere duplex replication and fill-in synthesis of the telomeric C-strand following telomerase action. CST also facilitates genome-wide replication recovery after HU-induced fork stalling by increasing origin firing. CTC1 and STN1 were originally isolated as a DNA polymerase α stimulatory factor. Here we explore how CST abundance affects recovery from drugs that cause different types of DNA damage and replication stress. We show that recovery from HU and aphidicolin induced replication stress is increased by CST over-expression. Elevated CST increases dNTP incorporation and origin firing after HU release and decreases the incidence of anaphase bridges and micronuclei after aphidicolin removal. While the frequency of origin firing after HU release is proportional to CST abundance, the number of cells entering S-phase to initiate replication is unchanged by CST overexpression or STN1 depletion. Instead the CST-related changes in origin firing take place in cells that were already in S-phase at the time of HU addition, indicating that CST modulates firing of late or dormant origins. CST abundance also influences cell viability after treatment with HU, aphidicolin, MMS and camptothecin. Viability is increased by elevated CST and decreased by STN1 depletion, indicating that endogenous CST levels are limiting. However, CST abundance does not affect viability after MMC treatment. Thus, CST facilitates recovery from many, but not all, forms of exogenous DNA damage. Overall our results suggest that CST is needed in stoichiometric amounts to facilitate re-initiation of DNA replication at repaired forks and/or dormant origins.     

SIRT1 Deacetylates TopBP1 and Modulates Intra-S-Phase Checkpoint and DNA Replication Origin Firing

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.     

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases.     

Multiple regulatory mechanisms to inhibit untimely initiation of DNA replication are important for stable genome maintenance

Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes.     

A model for DNA replication showing how dormant origins safeguard against replication fork failure

Replication origins are ‘licensed' for a single initiation event before entry into S phase; however, many licensed replication origins are not used, but instead remain dormant. The use of these dormant origins helps cells to survive replication stresses that block replication fork movement. Here, we present a computer model of the replication of a typical metazoan origin cluster in which origins are assigned a certain initiation probability per unit time and are then activated stochastically during S phase. The output of this model is in good agreement with experimental data and shows how inefficient dormant origins can be activated when replication forks are inhibited. The model also shows how dormant origins can allow replication to complete even if some forks stall irreversibly. This provides a simple explanation for how replication origin firing is regulated, which simultaneously provides protection against replicative stress while minimizing the cost of using large numbers of replication forks.     

Initiation of DNA Replication from Non-Canonical Sites on an Origin-Depleted Chromosome

Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.     

DNA replication origins fire stochastically in fission yeast

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.     

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.     

Prereplicative complexes assembled in vitro support origin‐dependent and independent DNA replication

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2‐7 helicase is first loaded into prereplicative complexes (pre‐RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre‐RCs assembled with purified proteins support complete and semi‐conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK). DDK phosphorylation of Mcm2‐7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin‐dependent in this system. These experiments indicate that Mcm2‐7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.