Biology subjects

Costa, A.

Publications and source records attributed to Costa, A..

At least 19 recordsLinked to original sources

bioRxiv2026

β-cyclocitral elicits rapid signaling distinct from the excess-light response in Arabidopsis

{beta}-Cyclocitral ({beta}-cc), a {beta}-carotene oxidation product, has been proposed as an early retrograde signal that primes protection against photooxidative stress. Its temporal relationship with photosynthetic acclimation, however, remains unresolved. Exogenous {beta}-cc elicited cytosolic calcium changes within minutes and induced the {beta}-cc-responsive pAER::LUC detoxification reporter within 40 min. We exposed Arabidopsis thaliana plants to 1500 mol photons m-2 s-1 under ventilation creating a high-irradiance regime without concomitant heat stress and with a relatively mild photooxidative outcome (RT-EL). Non-photochemical quenching increased, whereas PSII maximum quantum yield progressively decreased during RT-EL, with both responses approaching a plateau by 16 h. Volatile {beta}-cc pretreatment did not improve photosynthetic performance or NPQ dynamics, whereas endogenous {beta}-cc and {beta}-cyclocitric acid accumulated only after prolonged RT-EL exposure (16 h). By contrast, hormone profiling and RNA-seq revealed extensive {beta}-cc-dependent reprogramming that was largely opposite to the RT-EL transcriptional response. Together, these results reveal a kinetic separation between rapid {beta}-cc signaling and its endogenous production. Under RT-EL major photosynthetic adjustments precede detectable {beta}-cc accumulation, whereas {beta}-cc triggers cellular responses within minutes once present. We therefore propose that the timing of {beta}-cc action during excess light is determined primarily by the onset of substantial {beta}-carotene oxidation.

plant biology↗

bioRxiv2026

ER-located Ca2+ ATPase ACA2 regulates Ca2+ cytoplasmic pool linked to root hair growth in Arabidopsis thaliana

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is regulated by both intrinsic and environmental signals and is associated with the existence of a highly controlled cytoplasmic tip Ca{superscript 2} gradient, whose disruption impairs RH development. The molecular mechanisms underlying the Ca2+ homeostasis fine tuning and the Ca2+ organellar contributions to the cytoplasmic pool remain unclear. In the model plant Arabidopsis thaliana, many efflux routes are present, including those that employ Ca2+-pumps from the Autoinhibited Ca2+-ATPase (ACA) family. Here, we identified that the ER localized ACA2, and to a lower extent ACA7, are crucial ACAs required to control RH growth. By using genetically encoded Ca2+ biosensors we showed that Ca2+-dynamics are compromised in the aca2-2 mutant, having lower cytosolic Ca2+ concentration [Ca2+]cyt and growth rate, showing an altered homeostatic calcium setpoint compared to Col-0. Accordingly, the ACA2 mutation changed the dynamics of [Ca2+]cyt oscillations coupled to growth rate, inducing longer periods and more regular oscillations in the dominant high-frequency range (around 22 s), and slower oscillations (around 1 min) in the low-frequency range. Finally, expression of ACA2 with changes in four putative Ca2+ binding residues (ACA2{Delta}Ca2+) failed to rescue the RH growth phenotype in the aca2-2 mutant. Collectively, our findings indicate that ER-localized ACA2 and possibly ACA7 are crucial for modulating cytoplasmic Ca2+ signals, possibly composing a critical part of a negative feedback loop, and their absence leads to impairments in RH cell elongation.

plant biology↗

bioRxiv2026

Thalamic and cortical signals synergistically represent auditory prediction errors

Prediction errors (PEs) are commonly described as cortical signals generated within sensory hierarchies, but whether the thalamus participates in their encoding and transmission remains unclear. We recorded Local Field Potentials (LFP) from the medial and lateral geniculate nuclei and electrocorticography (ECoG) from multiple cortical regions in three awake cats during two auditory prediction tasks. Mutual information (MI) analyses revealed PE encoding in both thalamic and cortical signals. Co-information (co-I) analyses showed off-diagonal temporal synergy between early and later thalamic response components, consistent with an early response inducing a neural state change that shaped the informational content of subsequent activity. Multivariate co-information (MVCo-I) further revealed that thalamic and cortical population activity carried complementary PE information unavailable from either thalamic or cortical areas alone. These synergistic interactions were reliable across animals for violations of structured auditory sequences and weaker for repetition-based deviants. These findings show that auditory PEs are not simply relayed or duplicated across the thalamocortical hierarchy. Instead, they emerge through state-dependent transformations within the thalamus and complementary interactions between thalamic and cortical populations, identifying the thalamus as an active node of context-dependent PE processing.

neuroscience↗

bioRxiv2026

Habitat discontinuity and fidelity to migratory routes shape narwhal genetic structure and diversity

Rapid Arctic warming is reshaping marine ecosystems and altering the evolutionary trajectories of ice-associated species. Narwhals (Monodon monoceros) are Arctic endemics that are thought to be vulnerable to climate change. We present the first nuclear genomic assessment of narwhals across their distribution to evaluate population structure, demographic history, local adaptation, inbreeding and genetic load. Using genomes from 117 individuals, we identified three populations: Canadian Arctic Archipelago/West Greenland, Northeast Greenland/Svalbard and Southeast Greenland. Demographic reconstructions indicated low effective population size over at least 600,000 years, followed by population growth during the last glacial period. Our results suggest that population structure is maintained by habitat discontinuities and fidelity to migration routes. The latter may promote local adaptation, as genes related to long-term memory were found in regions putatively under selection in Canadian Arctic Archipelago/West Greenland narwhals, which undertake the longest migrations. Genome-wide diversity was uniformly low across populations. Inbreeding levels were inversely related to estimated population sizes. The small and rapidly declining Southeast Greenland population exhibited elevated recent inbreeding. Deleterious mutations were primarily masked in heterozygous genotypes, raising concerns for this population. Together, our results demonstrate that past climate, habitat discontinuities and migration fidelity jointly structure narwhal populations.

evolutionary biology↗

bioRxiv2026

Structure of the Pre-Initiation Complex Explains CMGE Biogenesis

Summary paragraphWhen cells enter S phase, bidirectional DNA replication is initiated through the kinase-regulated recruitment of three activators (Cdc45, GINS and Pol epsilon) to a duplex DNA-loaded double hexamer of MCM ATPases. Together these proteins form two CMGE helicases that establish divergent replication forks as they become separated1. To understand CMGE biogenesis, we reconstituted the pre-Initiation Complex with purified yeast proteins. The cryo-EM structure shows a set of firing factors caught in the act of assembling two symmetric CMGEs. We show how stepwise complex formation reshapes MCM in preparation for DNA opening and we explain how ATP promotes firing-factor ejection and CMGE maturation. While we find that Sld2 promotes GINS recruitment to MCM as expected, it also aids efficient separation of the CMGE dimer, and it is essential for lagging strand ejection from MCM. These findings have direct implications for our understanding of the metazoan Sld2 ortholog, RECQL4, pointing to a replication-fork establishment mechanism conserved across eukaryotes.

biochemistry↗

bioRxiv2026

Mcm10 braces converging replicative helicases to pull apart origin DNA

Eukaryotic DNA replication initiation requires controlled, temporally separated steps to preserve genome stability. The MCM replicative helicase is loaded on duplex DNA as an inactive double hexamer, which nucleates the assembly of dimeric Cdc45-MCM-GINS-Pol epsilon (dCMGE) replisomes. Mcm10 splits dCMGE into two, generating divergent replication forks, but the mechanism is unknown. Using ATPase-defective yeast MCM variants that slow origin melting, we captured five reaction intermediates that explain the structural mechanism. Two Mcm10 molecules bridge across the CMGE dimer, bracing two converging helicases. The restrained MCM motors pull apart the two DNA filaments, such that each lagging strand becomes ejected through the Mcm2-5 gate. Our reconstituted structures resemble the double CMG stabilized by metazoan DONSON, pointing to an origin DNA melting mechanism conserved across evolution.

biochemistry↗

bioRxiv2026

Modafinil Modulation of Oscillatory and Aperiodic Brain Activity in Rats Across Wakefulness and Subsequent Sleep

Cortical states shape sensory processing, decision-making, and behavior, and are characterized by distinct patterns of neuronal oscillatory activity. Pharmacological agents can modulate these states and their underlying electrophysiological signatures. Modafinil is a widely used wake-promoting agent and cognitive enhancer, but its effects on cortical oscillations are not fully understood. In this study, we evaluated the effect of modafinil on cortical activity during wakefulness and subsequent sleep. Ten male Wistar rats were implanted with electrocorticographic electrodes and recorded following oral administration of modafinil or its vehicle. Our results show that modafinil increased wakefulness in a dose-dependent manner, with doses of 200 and 300 mg/kg producing prolonged wake episodes and reducing hypnogram complexity. During wakefulness, modafinil enhanced gamma-band power in anterior cortices, an effect primarily driven by increases in the aperiodic rather than oscillatory components. Although no sleep rebound was observed, subsequent NREM sleep exhibited increased delta power, particularly in anterior regions, indicating heightened sleep pressure. In conclusion, our findings demonstrate that modafinil-induced wakefulness is associated with an enhanced aperiodic gamma band component at the anterior cortices in the EEG. Furthermore, modafinil modulates sleep homeostasis by enhancing NREM sleep intensity rather than extending its duration.

neuroscience↗

bioRxiv2026

Nutrient response strategies drive coastal range shifts of phytoplankton taxa

Coastal phytoplankton blooms are important drivers of regional and global marine primary production. Recently documented increases in coastal phytoplankton blooms in the first quarter of the 21st century may be a consequence in part of changing environmental conditions and have important ecological implications. Here, we explore coastal phytoplankton dynamics in Cape Cod Bay, MA, USA. We use a 20-year phytoplankton ecology dataset to identify potential drivers of the increasing prevalence of two regionally-rare phytoplankton taxa: a coccolithophore thought to thrive in the global open ocean, and a dinoflagellate genus with potentially toxic members. Using metatranscriptomics, we show that these minor phytoplankton taxa leverage unique strategies to gain a competitive advantage under nutrient limitation compared to traditionally dominant taxa and compared to a diatom taxon that became modestly more abundant over the study period. Our results highlight the ecological dynamics arising from long-term shifts in temperature and nutrient status in coastal ecosystems.

ecology↗

bioRxiv2025

Three-Dimensional Epigenome Roadmap of Human B-cell Differentiation Uncovers Mechanisms of Humoral Immunity and Oncogenesis

Human B-cell differentiation underlies humoral immunity, and its disruption leads to cancer, autoimmunity, and immunodeficiencies. However, the enhancer-based regulatory mechanisms governing this process remain poorly understood. To address this gap, we generated the first three-dimensional epigenomic roadmap of human B-cell differentiation in vivo. By profiling nine differentiation stages, we linked nearly half a million enhancers to their target genes through chromatin interactions, defined their activity states, and predicted bound transcription factors. We show that the acquisition of B-cell identity, distinct from common cellular processes, relies on large enhancer networks acting additively and synergistically to finely tune transcription. We further identify a 3D epigenetic priming mechanism underlying immune memory, whereby memory B cells retain primed DNA loops that enable faster responses to antigen re-exposure. Extending this roadmap to disease, we demonstrate that B-cell malignancies preserve enhancer signatures of their cell of origin while silencing distal tumor suppressor control. Finally, we uncover a previously unappreciated oncogenic mechanism in which intragenic deletions disrupt distal gene regulation via enhancer loss. Together, this resource provides a framework for understanding the genetic and epigenetic basis of humoral immunity and immune-related diseases.

genomics↗

bioRxiv2025

The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+ homeostasis

Calcium, as a cellular second messenger, is essential for plant growth. A tip-focused Ca2+ gradient in polarized cells is considered to drive cell expansion. The cell wall polysaccharide pectin is a major Ca2+ binding structure and Ca2+ homeostasis is influenced by the cell wall architecture. LRR-extensin (LRX) proteins are extracellular regulators of cell wall development that are anchored in the cell wall by their extensin domain. The extensin-less LRX1{Delta}E14 variant of the root hair-expressed LRX1 of Arabidopsis induces a dominant-negative effect resulting in aberrant root hairs. In an effort to identify the underlying mechanism of the root hair defect caused by LRX1{Delta}E14, we isolated a suppressor of dominant-negative effect mutant, sune42. It codes for the CATION CALCIUM EXCHANGER 4 (CCX4) that localizes to the Golgi apparatus and was shown to have Ca2+ transport activity. A detailed investigation of the Ca2+ dynamics revealed that LRX1{Delta}E14 coincides with a defect in tip-focused cytoplasmic Ca2+ oscillation, and this effect is alleviated by the sune42 mutation. Additionally, reducing Ca2+ availability influences the LRX1{Delta}E14-induced root hair defect. We conclude that sune42 suppresses the root hair defect in LRX1{Delta}E14 through modulating cytoplasmic Ca2+ dynamics, pointing at the importance of the Golgi apparatus for cellular Ca2+ homeostasis.

plant biology↗

bioRxiv2025

The Ca2+ Channel CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) regulates systemic wound signalling and immunity against Spodoptera herbivory

Insect herbivory elicits rapid and complex signalling responses in plants, including membrane depolarization, cytosolic Ca2+ elevation and activation of jasmonate-mediated defense pathways in local and systemic leaves. These early events are orchestrated by calcium-permeable channels and regulatory components that generate stimulus-specific cytosolic Ca2+ signatures. However, the molecular mechanisms that generate long-distance Ca2+ propagation and systemic immune activation on herbivory remain poorly defined. Here, we identify CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) as a novel plasma membrane-localized, phloem-expressed Ca2+ channel that is essential for systemic wound signalling and defense against lepidopteran pest, Spodoptera litura in Arabidopsis thaliana. CNGC13 is rapidly induced by wounding and herbivory, mediates cytosolic Ca2+ influx, and is required for efficient propagation of Ca2+ signals from wounded to distal leaves. Loss of CNGC13 compromises the thioglucosidase-dependent breakdown of aliphatic glucosinolates into aglucones/isothiocyanates within the vascular tissue, which are essential for wounding-induced systemic Ca2+ wave propagation. Consequently, cngc13 mutants display impaired systemic jasmonate accumulation in distal leaves, reduced glucosinolate levels and enhanced herbivore susceptibility. CNGC13 is also required for AtPep-induced Ca2+ elevation and Reactive Oxygen Species (ROS) signalling and interacts with the receptor kinase, PEPR2, implicating it in DAMP-mediated immune responses. Although CNGC13 and CNGC19 function cooperatively in herbivore defense, they do not physically interact, suggesting independent but converging roles. Our study identifies CNGC13 as a vasculature-localized Ca2+-permeable channel that functions downstream of PEPR2 receptor, to sustain systemic signalling, operating in a distinct yet converging pathway alongside known glutamate receptor-like channels during long-distance wound responses. SignificancePlants must detect and respond to insect herbivory rapidly by activating systemic immunity to trigger defense responses. Calcium (Ca2+) signalling plays a central role in this defense network, yet the identities and specific functions of Ca2+ channels involved in long-distance herbivory signalling remain poorly understood. Here, we identify the plasma membrane-localized channel CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) as a key integrator of mechanical damage perception, ricca factor-mediated systemic signalling, and jasmonate-dependent defense against Spodoptera herbivory in Arabidopsis. CNGC13 facilitates systemic Ca2+ wave propagation, activates jasmonate and glucosinolate biosynthesis, and promotes the perception of damage-associated molecular pattern (DAMP) molecules to confer herbivore resistance. This work uncovers a previously uncharacterized calcium channel that plays a crucial dual role in systemic wound signalling.

plant biology↗

bioRxiv2025

Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity

Stomata are natural pores through which plants exchange gases with the environment, mainly carbon dioxide and oxygen required for photosynthesis and respiration, as well as water vapor through evapotranspiration. However, they also serve as entry points for microbial pathogens such as Pseudomonas syringae pv. tomato (Pst) bacteria. To prevent microbe invasion, guard cells detect pathogens-associated molecular patterns (PAMPs), including the bacterial peptide flagellin (flg22), triggering stomatal closure. This study identifies hydrogen sulfide (H2S) and its cytosolic source L-CYSTEINE DESULFHIDRASE 1 (DES1), as key players in stomatal immunity. We demonstrate that H2S and DES1 are involved in flg22- and bacterial-induced responses, including stomatal closure and modulation of reactive oxygen species (ROS) production. We have found that knock out mutants in DES1 gene exhibits reduced susceptibility to Pst spray-inoculation and lower apoplastic and cytosolic H2O2 levels in response to flg22. Additionally, H2S independently induces cytosolic H2O2 levels in guard cells without requiring RBOHD activity. All together, these findings establish H2S and its source, DES1, as critical components of the stomatal immune response. One Sentence SummaryH2S and DES1 actively participate in flg22-induced stomatal closure modulating apoplastic and cytosolic ROS production.

plant biology↗

bioRxiv2024

Disparate demographic impacts of the Roman Colonization and the Migration Period in the Iberian Peninsula.

It has been unclear how the periods of Roman and later Germanic political control shaped the demography of the Iberian Peninsula and how Iberia differs in these respects from other parts of the Roman Empire. We report genome-wide data from 248 ancient individuals from the largely unsampled period 100-800 CE and co-analyze them with previously reported data. In the Roman era, we document profound demographic transformation, with an influx of people with ancestry from the Central and Eastern Mediterranean in all the areas under study and of North Africans, especially in central and southern Iberia. Germanic (Buri, Suebi, Vandals & Visigoths) and Sarmatian (Alans) took over political control beginning in the 5th century, and although we identify individuals with Germanic-associated ancestry at sites with Germanic-style ornaments and observe that such individuals were closely related across large distances as in the case of two siblings separated by 700 km, for Iberia as a whole, we observe high continuity with the previous Hispano-Roman population. The demographic patterns in Iberia contrast sharply with those in Britain, which showed the opposite pattern of little change in the Roman period followed by great change in the Migration period, and also from demographic patterns in the central Mediterranean where both periods were associated with profound transformation, raising broader questions about the forces that precipitated change over this time.

genomics↗

bioRxiv2024

Burning glass effect of water droplets triggers an ER-derived calcium response in the chloroplast stroma of Arabidopsis thaliana leaves

Plants require water and light for photosynthesis, but light, when focused by water droplets on leaves, can create high light intensity spots that are harmful to plants. As excessive light intensity can reduce growth or even induce cell death, it is vital for plants to detect and react to changes in light exposure and acclimate to high light stress. Ca2+ signaling was previously implicated in high light acclimation. However, the dynamics of free Ca2+ concentration in the chloroplast, the primary site of photosynthesis, or in the nucleus and in the cytoplasm, where transcription and translation for long-term acclimation occurs, remain unknown. Here we studied the dynamics and mechanism of the Ca2+ response to high light exposure. Focusing light through a glass bead to mimic water droplets triggered an increase of the free Ca2+ concentration in the chloroplast stroma of Arabidopsis thaliana. This finding was corroborated using established and newly developed genetically encoded calcium indicators, which revealed a biphasic increase in the stromal free Ca2+ concentration when exposed to varying intensities and qualities of light. Among photosynthetic by-products, reactive oxygen and lipophilic species in particular, have been implicated in high light stress acclimation. A H2O2 signature was induced, albeit with different dynamics than the Ca2+ response, while chemical inhibition of the photosynthetic electron transport points towards singlet oxygen as a potential culprit of the high light-induced increase in stromal free Ca2+ concentration. The observed dynamics differed from those of a heat-shock induced Ca2+ signature, although temperature had a positive effect on the Ca2+ response. Based on Ca2+ inhibitor treatments and the free Ca2+ concentration dynamics, we suggest that the high light-induced stromal Ca2+ is derived from the endoplasmic reticulum rather than from the cytoplasm. In conclusion, inspired by the burning glass effect of water droplets on leaves, we uncovered a Ca2+ response that implicates a novel mechanism for plants to acclimate to high light stress--a process that will become increasingly relevant in a changing climate.

plant biology↗

bioRxiv2024

Dopaminergic modulation of behavioral and electrocortical markers of interpersonal performance monitoring in Parkinson's Disease: insights from multivariate and univariate analyses

Effective interpersonal interaction necessitates constant monitoring and adaptation to others actions, a process known as interpersonal performance monitoring, which is influenced by the dopaminergic system and marked by specific electrocortical signatures. To explore the connection between deficits in interpersonal performance monitoring and altered neural markers, we assessed patients with Parkinsons Disease (PD) performing coordination tasks with a virtual partner (VP) under two conditions: on dopaminergic medication (PD ON) and after withdrawal (PD OFF). In Interactive trials, which required adaptation to the VPs actions, PD OFF performance was impaired compared to PD ON. EEG analysis revealed in PD OFF increased midfrontal Delta-Theta activity during Interactive trials. Higher Delta-Theta synchronization was associated with improved performance, suggesting compensatory mechanisms. Multivariate EEG analysis distinguished Interactive from Cued trials, especially in PD OFF. Our findings highlight dopamine s role in modulating electrocortical markers of interpersonal performance monitor, with significant implications for understanding and treating PD.

neuroscience↗

bioRxiv2024

Abiotic stress-induced chloroplast and cytosolic Ca2+ dynamics in the green alga Chlamydomonas reinhardtii

Calcium (Ca2+)-dependent signalling plays a well-characterized role in the perception and response mechanisms to environmental stimuli in plant cells. In the context of a constantly changing environment, it is fundamental to understand how crop yield and microalgal biomass productivity are affected by external factors. Ca2+ signalling is known to be important in different physiological processes in microalgae but many of these signal transduction pathways still need to be characterized. Here, the role of compartment-specific Ca2+ signalling was investigated in Chlamydomonas reinhardtii in response to environmental stressors such as nutrient availability, osmotic stress, temperature fluctuations and carbon sensing. An in vivo single-cell imaging approach was adopted to directly visualize signalling processes at the level of specific subcellular compartments, using Chlamydomonas reinhardtii lines expressing a genetically encoded ratiometric Ca2+ indicator. Hyper-osmotic shock caused cytosolic and chloroplast Ca2+ elevations, whereas high temperature and inorganic carbon availability primarily induced Ca2+ transients in the chloroplast. In contrast, hypo-osmotic stress only induced Ca2+ elevations in the cytosol. The results herein reported show that compartment-specific signalling pathways are likely to play an important role in the response of Chlamydomonas to these stimuli providing new understanding of the mechanisms exploited by microalgae to respond to specific natural conditions.

plant biology↗

bioRxiv2024

MCM Double Hexamer Loading Visualised with Human Proteins

Eukaryotic DNA replication begins with the loading of the MCM replicative DNA helicase as a head-to-head double hexamer (DH) at origins of DNA replication1-3. Our current understanding of how DH is assembled by the Origin Recognition Complex (ORC), CDC6 and CDT1 comes mostly from budding yeast. Here we characterise human DH (hDH) loading using biochemical reconstitution and cryo-electron microscopy with purified proteins. We show that hDH engages DNA differently from yeast (yDH), and generates [~]5 base pairs of unwound DNA at the interface between hexamers, as seen in hDH isolated from cells4. We identify several differences from yeast in the order of factor recruitment and dependencies during hDH assembly. Unlike yeast5-8, the ORC6 subunit of ORC is not essential for initial MCM recruitment or hDH loading, but contributes to an alternative hDH assembly pathway requiring an intrinsically disordered region (IDR) in ORC1, which may work through a novel MCM-ORC (hMO*) intermediate. Our work presents a detailed view of how DHs are assembled in an organism utilising sequence-independent replication origins, it provides further evidence for diversity in eukaryotic DH assembly mechanisms9, and it represents the first step toward reconstitution of DNA replication initiation with purified human proteins.

biochemistry↗

bioRxiv2024

Cell Cycle Regulation has Shaped Budding Yeast Replication Origin Structure and Function

Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

biochemistry↗