Transcription-Replication Conflicts as a Source of Genome Instability.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

Hold on Tight: Lagging-Strand DNA Polymerases Synthesize Multiple Okazaki Fragments without Letting Go.

Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair.

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.

Use of nominal group technique methods in the virtual setting: A reflective account and recommendations for practice.

Nominal group technique methods involve the use of structured activities within groups comprised of purposefully selected stakeholders (nominal groups), with the broad aim of achieving a level of consensus and prioritising information. In this paper, we will report how we facilitated nominal groups, using Microsoft Teams, to prioritise content for a theory-based behaviour change intervention to improve responses to clinically deteriorating patients. Our methods incorporated development and piloting of research materials, facilitation of online nominal groups with different stakeholders, and a structured approach to ranking behaviour change strategies. Practical suggestions are offered based on our experience of using this method in a virtual context.

A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.

High-throughput metabolomics predicts drug-target relationships for eukaryotic proteins.

Chemical probes are important tools for understanding biological systems. However, because of the huge combinatorial space of targets and potential compounds, traditional chemical screens cannot be applied systematically to find probes for all possible druggable targets. Here, we demonstrate a novel concept for overcoming this challenge by leveraging high-throughput metabolomics and overexpression to predict drug-target interactions. The metabolome profiles of yeast treated with 1,280 compounds from a chemical library were collected and compared with those of inducible yeast membrane protein overexpression strains. By matching metabolome profiles, we predicted which small molecules targeted which signaling systems and recovered known interactions. Drug-target predictions were generated across the 86 genes studied, including for difficult to study membrane proteins. A subset of those predictions were tested and validated, including the novel targeting of GPR1 signaling by ibuprofen. These results demonstrate the feasibility of predicting drug-target relationships for eukaryotic proteins using high-throughput metabolomics.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Linking stem rehydration kinetics to hydraulic traits using a novel method and mechanistic model.

BACKGROUND: Despite the recognized importance of hydraulic capacitance as a mechanism used by plants to maintain hydraulic functioning during high transpiration, characterizing the dynamics of capacitance remains a challenge. METHODS: We used a novel 'two-balance method' to investigate relationships between stem rehydration kinetics and other hydraulic traits in multiple tree species, and we developed a model to explore stem rehydration kinetics further. KEY RESULTS: We found that: (1) rehydration time constants and the amount of water uptake occurring during rehydration differed significantly across species; (2) time constants did not change with declining water potential (Ψ), while water uptake increased at lower Ψ in some species; (3) longer time constants were associated with lower wood density, higher capacitance and less negative stem pressures causing 50 % loss of hydraulic conductivity (P50); (4) greater water uptake occurred in stems with lower wood density and less negative P50 values; and (5) the model could estimate the total hydraulic resistance of the rehydration path, which cannot be measured directly. CONCLUSIONS: Overall, the two-balance method can be used to examine rehydration dynamics quickly and thoroughly in detached woody stems. This method has the potential to improve our understanding of how capacitance functions across tree species, which is an often-overlooked component of whole-plant hydraulics.

Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation.

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.

Development and psychometric evaluation of the Attitudes Towards Recognising Early and Noticeable Deterioration (ATREND) scale.

AIMS AND OBJECTIVES: To develop and evaluate the psychometric properties of an instrument that measures nurses' Attitudes Towards Recognising Early and Noticeable Deterioration (ATREND). BACKGROUND: General ward nurses play an important role in recognising patient deterioration. However, their attitudes towards early recognition of clinical deterioration have not been adequately explored due to the lack of a valid and reliable scale. DESIGN: An instrument development and validation study. METHODS: A three-phase structure that followed the STROBE checklist was used: (1) item generation, (2) content and face validity assessment and (3) psychometric properties evaluation. The scale items were developed based on a comprehensive literature review and content validity assessment by 15 international experts from five countries. The psychometric properties of the ATREND scale were tested on 434 registered nurses, with retest evaluations (n = 100) at two hospitals. Exploratory and confirmatory factor analyses were used to examine the factor structure of the scale. The scale was also evaluated for its internal consistency, test-retest reliability and convergent validity. RESULTS: The scale's content validity was 0.95. A 3-factor solution was identified from the final 11 items: (1) beliefs about importance of patient observation, (2) use of broader patient assessment skills and (3) confidence in recognising clinical deterioration. The internal consistency reliability of the scale was supported with an acceptable Cronbach's alpha value of 0.745. Test-retest reliability of the scale was excellent, with an intraclass correlation coefficient of 0.825. The ATREND scale shows evidence of good convergent validity. CONCLUSION: The final 11-item ATREND scale demonstrates adequate initial evidence of reliability and validity for use in acute ward settings. RELEVANCE TO CLINICAL PRACTICE: Nursing educators and clinicians may use this scale to assess ward nurses' attitudes and practices towards early recognition of clinical deterioration and then enhance their competencies and behaviours in the recognition of clinical deterioration.

Collaborative practice among general ward staff on escalating care in clinical deterioration: A systematic review.

AIM: To understand the issues surrounding collaborative practice and collaboration experiences among general ward staff in the escalation of care for clinically deteriorating patients. DESIGN: A systematic synthesis without meta-analysis. REVIEW METHODS: Seven electronic databases (CINAHL, Cochrane, Embase, PsycINFO, PubMed, Scopus and ProQuest Theses and Dissertations) were searched from their inception to 30 April 2022. Two reviewers independently screened titles, abstracts and full text for eligibility. The critical appraisal skill programme, Joanna Briggs Institute checklist for analytical cross-sectional studies and mixed methods appraisal tool were used to appraise the quality of the included studies. Both quantitative and qualitative research data were extracted, analysed and then synthesised using the data-based convergent qualitative synthesis approach. This review adhered to the Synthesis without meta-analysis (SWiM) reporting guidelines. RESULTS: A total of 17 studies were included. Two themes and six sub-themes were generated: (1) intraprofessional factors-inadequate handover, workload and mutual support, raising and acting on concerns, and seeking help from seniors and (2) interprofessional factors-differences in communication styles, and hierarchical approach versus interpersonal relationships. CONCLUSIONS: This systematic review highlights the need to address the intra- and interprofessional issues surrounding collaborative practice in escalation of care among general ward staff. IMPLICATIONS FOR THE PROFESSION: Findings from this review will inform healthcare leaders and educators on the development of relevant strategies and multi-disciplinary training to foster effective teamwork among nurses and doctors, with the goal of improving the escalation of care for patients with clinical deterioration. NO PATIENT OR PUBLIC CONTRIBUTION: This systematic review did not directly involve patient or public contribution to the manuscript.

Strain Variation Can Significantly Modulate the miRNA Response to Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-transmitted virus that has emerged as a major public health concern due to its association with neurological disorders in humans, including microcephaly in fetuses. ZIKV infection has been shown to alter the miRNA profile in host cells, and these changes can contain elements that are proviral, while others can be antiviral in action. In this study, the expression of 22 miRNAs in human A549 cells infected with two different ZIKV isolates was investigated. All of the investigated miRNAs showed significant changes in expression at at least one time point examined. Markedly, 18 of the miRNAs examined showed statistically significant differences in expression between the two strains examined. Four miRNAs (miR-21, miR-34a, miR-128 and miR-155) were subsequently selected for further investigation. These four miRNAs were shown to modulate antiviral effects against ZIKV, as downregulation of their expression through anti-miRNA oligonucleotides resulted in increased virus production, whereas their overexpression through miRNA mimics reduced virus production. However, statistically significant changes were again seen when comparing the two strains investigated. Lastly, candidate targets of the miRNAs miR-34a and miR-128 were examined at the level of the mRNA and protein. HSP70 was identified as a target of miR-34a, but, again, the effects were strain type-specific. The two ZIKV strains used in this study differ by only nine amino acids, and the results highlight that consideration must be given to strain type variation when examining the roles of miRNAs in ZIKV, and probably other virus infections.

A practical approach to establishing a critical care outreach service: An expert panel research design.

BACKGROUND: For over two decades, nurse-led critical care outreach services have improved the recognition, response, and management of deteriorating patients in general hospital wards, yet variation in terms, design, implementation, and evaluation of such services continue. For those establishing a critical care outreach service, these factors make the literature difficult to interpret and translate to the real-world setting. AIM: The aim of this study was to provide a practical approach to establishing a critical care outreach service in the hospital setting. METHOD: An international expert panel of clinicians, managers, and academics with experience in implementing, developing, operationalising, educating, and evaluating critical care outreach services collaborated to synthesise evidence, experience, and clinical judgment to develop a practical approach for those establishing a critical care outreach service. A rapid review of the literature identified publications relevant to the study. A modified Delphi technique was used to achieve expert panel consensus particularly in areas where insufficient published literature or ambiguities existed. FINDINGS: There were 502 publications sourced from the rapid review, of which 104 were relevant and reviewed. Using the modified Delphi technique, the expert panel identified five key components needed to establish a critical care outreach service: (i) approaches to service delivery, (ii) education and training, (iii) organisational engagement, (iv) clinical governance, and (v) monitoring and evaluation. CONCLUSION: An expert panel research design successfully synthesised evidence, experience, and clinical judgement to provide a practical approach for those establishing a critical care outreach service. This method of research will likely be valuable in other areas of practice where terms are used interchangeably, and the literature is diverse and lacking a single approach to practice.

Hydroscapes, hydroscape plasticity and relationships to functional traits and mesophyll photosynthetic sensitivity to leaf water potential in Eucalyptus species.

The isohydric-anisohydric continuum describes the relative stringency of stomatal control of leaf water potential (ψleaf ) during drought. Hydroscape area (HA)-the water potential landscape over which stomata regulate ψleaf -has emerged as a useful metric of the iso/anisohydric continuum because it is strongly linked to several hydraulic, photosynthetic and structural traits. Previous research on HA focused on broad ecological patterns involving several plant clades. Here we investigate the relationships between HA and climatic conditions and functional traits across ecologically diverse but closely related species while accounting for phylogeny. Across a macroclimatic moisture gradient, defined by the ratio of mean annual precipitation to mean annual pan evaporation (P/Ep ), HA decreased with increased P/Ep across 10 Eucalyptus species. Greater anisohydry reflects lower turgor loss points and greater hydraulic safety, mirroring global patterns. Larger HA coincides with mesophyll photosynthetic capacity that is more sensitive to ψleaf . Hydroscapes exhibit little plasticity in response to variation in water supply, and the extent of plasticity does not vary with P/Ep of native habitats. These findings strengthen the case that HA is a useful metric for characterizing drought tolerance and water-status regulation.

Scaling the leaf length-times-width equation to predict total leaf area of shoots.

BACKGROUND AND AIMS: An individual plant consists of different-sized shoots, each of which consists of different-sized leaves. To predict plant-level physiological responses from the responses of individual leaves, modelling this within-shoot leaf size variation is necessary. Within-plant leaf trait variation has been well investigated in canopy photosynthesis models but less so in plant allometry. Therefore, integration of these two different approaches is needed. METHODS: We focused on an established leaf-level relationship that the area of an individual leaf lamina is proportional to the product of its length and width. The geometric interpretation of this equation is that different-sized leaf laminas from a single species share the same basic form. Based on this shared basic form, we synthesized a new length-times-width equation predicting total shoot leaf area from the collective dimensions of leaves that comprise a shoot. Furthermore, we showed that several previously established empirical relationships, including the allometric relationships between total shoot leaf area, maximum individual leaf length within the shoot and total leaf number of the shoot, can be unified under the same geometric argument. We tested the model predictions using five species, all of which have simple leaves, selected from diverse taxa (Magnoliids, monocots and eudicots) and from different growth forms (trees, erect herbs and rosette herbs). KEY RESULTS: For all five species, the length-times-width equation explained within-species variation of total leaf area of a shoot with high accuracy (R2 > 0.994). These strong relationships existed despite leaf dimensions scaling very differently between species. We also found good support for all derived predictions from the model (R2 > 0.85). CONCLUSIONS: Our model can be incorporated to improve previous models of allometry that do not consider within-shoot size variation of individual leaves, providing a cross-scale linkage between individual leaf-size variation and shoot-size variation.

Selecting intervention content to target barriers and enablers of recognition and response to deteriorating patients: an online nominal group study.

BACKGROUND: Patients who deteriorate in hospital wards without appropriate recognition and/or response are at risk of increased morbidity and mortality. Track-and-trigger tools have been implemented internationally prompting healthcare practitioners (typically nursing staff) to recognise physiological changes (e.g. changes in blood pressure, heart rate) consistent with patient deterioration, and then to contact a practitioner with expertise in management of acute/critical illness. Despite some evidence these tools improve patient outcomes, their translation into clinical practice is inconsistent internationally. To drive greater guideline adherence in the use of the National Early Warning Score tool (a track-and-trigger tool used widely in the United Kingdom and parts of Europe), a theoretically informed implementation intervention was developed (targeting nursing staff) using the Theoretical Domains Framework (TDF) version 2 and a taxonomy of Behaviour Change Techniques (BCTs). METHODS: A three-stage process was followed: 1. TDF domains representing important barriers and enablers to target behaviours derived from earlier published empirical work were mapped to appropriate BCTs; 2. BCTs were shortlisted using consensus approaches within the research team; 3. shortlisted BCTs were presented to relevant stakeholders in two online group discussions where nominal group techniques were applied. Nominal group participants were healthcare leaders, senior clinicians, and ward-based nursing staff. Stakeholders individually generated concrete strategies for operationalising shortlisted BCTs ('applications') and privately ranked them according to acceptability and feasibility. Ranking data were used to drive decision-making about intervention content. RESULTS: Fifty BCTs (mapped in stage 1) were shortlisted to 14 (stage 2) and presented to stakeholders in nominal groups (stage 3) alongside example applications. Informed by ranking data from nominal groups, the intervention was populated with 12 BCTs that will be delivered face-to-face, to individuals and groups of nursing staff, through 18 applications. CONCLUSIONS: A description of a theory-based behaviour change intervention is reported, populated with BCTs and applications generated and/or prioritised by stakeholders using replicable consensus methods. The feasibility of the proposed intervention should be tested in a clinical setting and the content of the intervention elaborated further to permit replication and evaluation.

The Roles of Mitophagy and Autophagy in Ineffective Erythropoiesis in ß-Thalassemia.

ß-Thalassemia is one of the most common genetically inherited disorders worldwide, and it is characterized by defective ß-globin chain synthesis leading to reduced or absent ß-globin chains. The excess α-globin chains are the key factor leading to the death of differentiating erythroblasts in a process termed ineffective erythropoiesis, leading to anemia and associated complications in patients. The mechanism of ineffective erythropoiesis in ß-thalassemia is complex and not fully understood. Autophagy is primarily known as a cell recycling mechanism in which old or dysfunctional proteins and organelles are digested to allow recycling of constituent elements. In late stage, erythropoiesis autophagy is involved in the removal of mitochondria as part of terminal differentiation. Several studies have shown that autophagy is increased in earlier erythropoiesis in ß-thalassemia erythroblasts, as compared to normal erythroblasts. This review summarizes what is known about the role of autophagy in ß-thalassemia erythropoiesis and shows that modulation of autophagy and its interplay with apoptosis may provide a new therapeutic route in the treatment of ß-thalassemia. Literature was searched and relevant articles were collected from databases, including PubMed, Scopus, Prospero, Clinicaltrials.gov, Google Scholar, and the Google search engine. Search terms included: ß-thalassemia, ineffective erythropoiesis, autophagy, novel treatment, and drugs during the initial search. Relevant titles and abstracts were screened to choose relevant articles. Further, selected full-text articles were retrieved, and then, relevant cross-references were scanned to collect further information for the present review.

Mesophyll photosynthetic sensitivity to leaf water potential in Eucalyptus: a new dimension of plant adaptation to native moisture supply.

Photosynthetic sensitivity to drought is a fundamental constraint on land-plant evolution and ecosystem function. However, little is known about how the sensitivity of photosynthesis to nonstomatal limitations varies among species in the context of phylogenetic relationships. Using saplings of 10 Eucalyptus species, we measured maximum CO2 -saturated photosynthesis using A-ci curves at several different leaf water potentials (ψleaf ) to quantify mesophyll photosynthetic sensitivity to ψleaf (MPS), a measure of how rapidly nonstomatal limitations to carbon uptake increase with declining ψleaf . MPS was compared to the macroclimatic moisture availability of the species' native habitats, while accounting for phylogenetic relationships. We found that species native to mesic habitats have greater MPS but higher maximum photosynthetic rates during non-water-stressed conditions, revealing a trade-off between maximum photosynthesis and drought sensitivity. Species with lower turgor loss points have lower MPS, indicating coordination among photosynthetic and water-relations traits. By accounting for phylogenetic relationships among closely related species, we provide the first compelling evidence that MPS in Eucalyptus evolved in an adaptive fashion with climatically determined moisture availability, opening the way for further study of this poorly explored dimension of plant adaptation to drought.

Mistletoes and their eucalypt hosts differ in the response of leaf functional traits to climatic moisture supply.

Trade-offs between photosynthesis and the costs of resource capture inform economic strategies of plants across environmental gradients and result in predictable variation in leaf traits. However, understudied functional groups like hemiparasites that involve dramatically different strategies for resource capture may have traits that deviate from expectations. We measured leaf traits related to gas exchange in mistletoes and their eucalypt hosts along a climatic gradient in relative moisture supply, measured as the ratio of precipitation to pan evaporation (P/Ep), in Victoria, Australia. We compared traits for mistletoes vs. hosts as functions of relative moisture supply and examined trait-trait correlations in both groups. Eucalypt leaf traits responded strongly to decreasing P/Ep, consistent with economic theory. Leaf area and specific leaf area (SLA) decreased along the P/Ep gradient, while C:N ratio, leaf thickness, N per area, and δ13C all increased. Mistletoes responded overall less strongly to P/Ep based on multivariate analyses; individual traits sometimes shifted in parallel with those of hosts, but SLA, leaf thickness, and N per area showed no significant change across the gradient. For mistletoes, leaf thickness was inversely related to leaf dry matter content (LDMC), with no relationship between SLA and mass-based N. In mistletoes, reduced costs of transpiration (reflecting their lack of roots) and abundant succulent leaf tissue help account for observed differences from their eucalypt hosts. Trait-based analysis of atypical functional types such as mistletoes help refine hypotheses based on plant economics and specialized adaptations to resource limitation.

A PP1-PP2A phosphatase relay controls mitotic progression.

The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.