Biology subjects

Cook, J. G.

Publications and source records attributed to Cook, J. G..

18 recordsLinked to original sources

bioRxiv2026

Molecular control of replication licensing speed by c-MYC

A critical prerequisite for DNA replication is genome-wide origin licensing, a process tightly restricted to the G1 cell cycle phase. This restriction means that short G1 cells must license quickly or risk incomplete replication and genome instability, but mechanisms to increase licensing speed are largely unknown. Using quantitative single cell approaches, we identify here a non-canonical role for c-MYC as a potent origin licensing accelerator. Importantly, a point mutation separates c-MYC-induced rapid licensing from the well-known c-MYC gene expression function. c-MYC accelerates licensing through the TRRAP lysine acetyltransferase adapter and GCN5 acetyltransferase. In addition c-MYC has the unexpected ability to induce widespread linker histone H1 eviction. Moreover, directly reducing chromatin-bound histone H1 is sufficient to accelerate licensing. In addition, c-MYC stimulates licensing in heterochromatin, a typically slow-licensing genome compartment. We suggest that global chromatin opening promotes rapid licensing when G1 phase is short such as during early development, rapid progenitor cell proliferation, and adaptation to oncogene activation.

molecular biology↗

bioRxiv2026

ELDR-Glo, a biosensor for cell age and quiescence depth

Fluorescent reporters are powerful tools for revealing intercellular heterogeneity among proliferating cells. However, there are few tools to analyze differences among quiescent (G0) cells, though such differences are relevant for development, tissue maintenance, and cancer cell behavior. Quiescence heterogeneity, also known as quiescence depth, typically correlates with time after cell cycle arrest, yet directly measuring cell age is not feasible for all cell types or most tissues. Here, we describe ELDR-Glo, a genetically-encoded fluorescent biosensor that estimates relative cell age, or time since the last cell cycle. The biosensor integrates replication-coupled degradation in S phase with a slow-maturing mCherry and a normalization module. We demonstrate that ELDR-Glo signal correlates with true cell age by both live-cell imaging and in fixed cells. ELDR-Glo distinguishes early and late G0 cells and functions as a relative quiescence depth reporter in situ. The biosensor is compatible with multiplexed immunofluorescence and flow cytometry. ELDR-Glo provides a unique and scalable tool to investigate cell proliferation control. TeaserA fluorescent biosensor to distinguish cells of different ages and thus, likelihood of returning to proliferation from quiescence.

molecular biology↗

bioRxiv2024

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of proliferative tip cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

cell biology↗

bioRxiv2023

APC/C prevents non-canonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear if APC/C maintains all types of arrest. Here by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological CDK4/6 inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves cyclin-dependent kinases acting in an atypical order to inactivate RB-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability. SignificanceAppropriate stable cell cycle arrest is critical to prevent cancer. However, it is not well-understood how cells maintain arrest. It is known that arrest requires repressing proliferation-stimulating genes, but the role of targeted protein degradation is unclear. This work demonstrates that continuous degradation of cyclin A through the action of the anaphase promoting complex/cyclosome (APC/C) is required to maintain arrest induced by a cancer drug that blocks cell cycle kinase enzymes. APC/C activity is required to prevent cell cycle re-entry. Impaired APC/C activity causes arrest bypass, inefficient DNA replication, and ultimately long-term proliferation defects. These results suggest that the activity level of the APC/C in tumors may profoundly influence the response to drugs that target cell cycle kinases.

cell biology↗

bioRxiv2023

HES1 and ID3 temporally regulate p27 levels and endothelial cell flow-mediated quiescence depth

BackgroundEndothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. MethodsPrimary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time to cell cycle re-entry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA seq analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. ResultsArterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous-flow exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. ConclusionsEndothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence mis-regulation that leads to vascular dysfunction and disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/544403v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1137a1forg.highwire.dtl.DTLVardef@1b6bf1dorg.highwire.dtl.DTLVardef@bd4965org.highwire.dtl.DTLVardef@11dbb9b_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIDifferent quiescence stimuli lead to distinct transcriptional and functional quiescence profiles in endothelial cells C_LIO_LIp27 is required for endothelial cell quiescence and depth is temporally regulated in a flow stimulus-dependent manner that correlates with p27 levels and flow-regulated repressors HES1 and ID3 C_LIO_LIp27 is expressed in endothelial cells according to flow magnitude in vivo and is functionally required for cell cycle regulation and sprouting in vivo C_LI

cell biology↗

bioRxiv2022

The molecular architecture of cell cycle arrest

The cellular decision governing the transition between proliferative and arrested states is crucial to the development and function of every tissue. While the molecular mechanisms that regulate the proliferative cell cycle are well established, we know comparatively little about what happens to cells as they diverge into cell cycle arrest. We performed hyperplexed imaging of 49 cell cycle effectors to obtain a map of the molecular architecture that governs cell cycle exit and progression into reversible ("quiescent") and irreversible ("senescent") arrest states. Using this map, we found multiple points of divergence from the proliferative cell cycle; identified stress-specific states of arrest; and resolved the molecular mechanisms governing these fate decisions, which we validated by single-cell, time-lapse imaging. Notably, we found that cells can exit into senescence from either G1 or G2; however, both subpopulations converge onto a single senescent state with a G1-like molecular signature. Cells can escape from this "irreversible" arrest state through the upregulation of G1 cyclins. This comprehensive map provides a first glimpse of the overall organization of cell proliferation and arrest.

cell biology↗

bioRxiv2021

Quantitative Profiling of Adaptation to Cyclin E Overproduction

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

cell biology↗

bioRxiv2021

The Consequences of Differential Origin Licensing Dynamics

MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

molecular biology↗

bioRxiv2021

The structure of the human cell cycle

The human cell cycle is conventionally depicted as a five-phase model consisting of four proliferative phases (G1, S, G2, M) and a single state of arrest (G0). However, recent studies show that individual cells can take different paths through the cell cycle and exit into distinct arrest states, thus necessitating an update to the canonical model. We combined time lapse microscopy, highly multiplexed single cell imaging and manifold learning to determine the underlying "structure" of the human cell cycle under multiple growth and arrest conditions. By visualizing the cell cycle as a complete biological process, we identified multiple points of divergence from the proliferative cell cycle into distinct states of arrest, revealing multiple mechanisms of cell cycle exit and re-entry and the molecular routes to senescence, endoreduplication and polyploidy. These findings enable the visualization and comparison of alternative cell cycles in development and disease. One-sentence summaryA systems-level view of single-cell states reveals the underlying architecture of the human cell cycle

cell biology↗

bioRxiv2021

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

cancer biology↗

bioRxiv2020

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

systems biology↗

bioRxiv2019

Chronic Mcm10 deficiency causes defects in telomere maintenance in human cells

Minichromosome maintenance protein 10 (Mcm10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 mutations in patients with distinctive but overlapping clinical phenotypes - natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of Mcm10-associated disease, we modeled these mutations in human cell lines. Mcm10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of Mcm10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in Mcm10-deficient cells require endonucleolytic processing by Mus81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic mutations in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes in both NKD and CM patients.

molecular biology↗

bioRxiv2018

Phosphoregulation of Cdt1 in G2 and M phases prevents re-replication independently of Geminin

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. 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 hyperphosphorylates 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, we provide evidence that protein phosphatase 1-dependent 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 SummaryThe 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 cant 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, but rather distinct mechanisms are dominant in different cell cycle phases. 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.

molecular biology↗

bioRxiv2018

Precise detection of S phase onset reveals decoupled G1/S transition events

The eukaryotic cell division cycle is the process by which cells duplicate their genomes and proliferate. Transitions between sequential cell cycle phases are tightly orchestrated to ensure precise and efficient cell cycle progression. Interrogating molecular events at these transitions is important for understanding normal and pathological cell proliferation and mechanisms that ensure genome stability. A popular fluorescent reporter system known as \"FUCCI\" has been widely adopted for identifying cell cycle phases. Using time-lapse fluorescence microscopy, we quantitatively analyzed the dynamics of the FUCCI reporters relative to the transitions into and out of S phase. Although the original reporters reflect the E3 ubiquitin ligase activities for which they were designed, SCFSkp2 and APCCdh1, their dynamics are significantly and variably offset from actual S phase boundaries. To precisely mark these transitions, we generated and thoroughly validated a new reporter containing a PCNA-interacting protein degron whose oscillations are directly coupled to the process of DNA replication itself. We combined this reporter with the geminin-based APCCdh1 reporter to create \"PIP-FUCCI.\" PIP degron reporter dynamics closely correlate with S phase transitions irrespective of reporter expression levels. Using PIP-FUCCI, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry, though delayed APCCdh1 inactivation correlates with longer S phase. Our results illustrate the benefits of precise delineation of cell cycle phase boundaries for uncovering the sequences of molecular events at critical cell cycle transitions.

cell biology↗

bioRxiv2018

Cdt1 variants reveal unanticipated aspects of interactions with Cyclin/CDK and MCM important for normal genome replication.

The earliest step in DNA replication is origin licensing which is the DNA loading of MCM helicase complexes. The Cdt1 protein is essential for MCM loading during G1 phase of the cell cycle, yet the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than WT Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex position the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both Cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live cell imaging analysis demonstrated no change in stability of this variant however. Instead, we propose that Cyclin A/CDK inhibits Cdt1 licensing function independently of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

molecular biology↗

bioRxiv2017

The Cell Cycle Browser: an interactive tool for visualizing, simulating, and perturbing cell cycle progression

SUMMARYThe cell cycle is driven by precise temporal coordination among many molecular activities. To understand and explore this process, we developed the Cell Cycle Browser (CCB), an interactive web interface based on real-time reporter data collected in proliferating human cells. This tool facilitates visualizing, simulating, and predicting the outcomes of perturbing cell cycle parameters. Time-series traces from individual cells can be combined to build a multi-layered timeline of molecular activities. Users can simulate the cell cycle using computational models that capture the dynamics of molecular activities and phase transitions. By adjusting individual expression levels and strengths of molecular relationships, users can predict effects on the cell cycle. Virtual assays, such as growth curves and flow cytometry, provide familiar outputs to compare cell cycle behaviors for data and simulations. The CCB serves to unify our understanding of cell cycle dynamics and provides a platform for generating hypotheses through virtual experiments.\n\nHIGHLIGHTSO_LIUsers can stack and align single-cell traces for different molecular reporters\nC_LIO_LIComputational models with adjustable parameters simulate cell cycle progression\nC_LIO_LIVirtual growth curves and flow cytometry assays predict cell cycle behaviors\nC_LI

systems biology↗

bioRxiv2017

Rapid DNA Replication Origin Licensing Protects Stem Cell Pluripotency

Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

cell biology↗

bioRxiv2017

DNA Damage Checkpoint Dynamics Drive Cell Cycle Phase Transitions

DNA damage checkpoints are cellular mechanisms that protect the integrity of the genome during cell cycle progression. In response to genotoxic stress, these checkpoints halt cell cycle progression until the damage is repaired, allowing cells enough time to recover from damage before resuming normal proliferation. Here, we investigate the temporal dynamics of DNA damage checkpoints in individual proliferating cells by observing cell cycle phase transitions following acute DNA damage. We find that in gap phases (G1 and G2), DNA damage triggers an abrupt halt to cell cycle progression in which the duration of arrest correlates with the severity of damage. However, cells that have already progressed beyond a proposed \"commitment point\" within a given cell cycle phase readily transition to the next phase, revealing a relaxation of checkpoint stringency during later stages of certain cell cycle phases. In contrast to G1 and G2, cell cycle progression in S phase is significantly less sensitive to DNA damage. Instead of exhibiting a complete halt, we find that increasing DNA damage doses leads to decreased rates of S-phase progression followed by arrest in the subsequent G2. Moreover, these phase-specific differences in DNA damage checkpoint dynamics are associated with corresponding differences in the proportions of irreversibly arrested cells. Thus, the precise timing of DNA damage determines the sensitivity, rate of cell cycle progression, and functional outcomes for damaged cells. These findings should inform our understanding of cell fate decisions after treatment with common cancer therapeutics such as genotoxins or spindle poisons, which often target cells in a specific cell cycle phase.

systems biology↗