Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.

cGAS surveillance of micronuclei links genome instability to innate immunity.

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Supporting farmer wellbeing: exploring a potential role for farm advisory staff.

INTRODUCTION: Farmers in Ireland experience poor health outcomes and are considered 'hard to reach'. Agricultural advisors ('advisors') are uniquely positioned to support and signpost farmers on health issues. This paper explores the acceptability and terms of reference of a potential health role for advisors, and offers key recommendations for developing a bespoke farmers' health training programme. METHODS: Following ethical approval, eleven focus groups (n=26 female, n=35 male, age-range 20s-70s) were conducted with farmers (n=4), advisors (n=4), farming organisations (n=2) and farmers' 'significant others' (n=1). Utilizing Thematic Content Analysis, transcripts were coded iteratively and emerging themes were grouped into primary and subthemes. RESULTS: Our analysis identified three themes. 'Scope and acceptability of a potential health role for advisors' examines how participants envision and are receptive to such a role. 'Roles, responsibilities and boundaries' considers both a health promotion and 'health connector' advisory role - normalising health conversations and signposting farmers to services/supports. Finally, 'trouble-shooting potential obstacles to advisors assuming a health role' reflects on the barriers that may impede advisors capacity or potential to have a broader health role. DISCUSSION: Within the context of the stress process theory, findings provide unique insights into how advisory can mediate stress and contribute to farmers' health and wellbeing. Finally, findings have important implications for potentially extending the reach of training to other aspects of farming support services (eg agri-banking, agri-business, veterinary services etc.), as well as serving as a springboard for the development of similar initiatives in other jurisdictions.

Barriers and facilitators to implementing community-based physical activity interventions: a qualitative systematic review.

BACKGROUND: Over the past decade several physical activity (PA) interventions have been shown to be efficacious in a controlled research setting, however there is a continued lack of evidence for how to successfully implement these PA interventions in real-world settings such as the community. This review aims to explore the barriers and facilitators that affect the implementation of community-based PA interventions and make recommendations to improve implementation from the included studies. METHODS: A systematic literature search of EBSCOhost, Scopus, PUBMED and Web of Science was conducted to identify articles that reported qualitative data on the implementation factors of community-based interventions where PA was a primary outcome. Data were extracted using the Consolidated Framework for Implementation Research (CFIR) as a guide. Implementation factors and recommendations were then mapped onto the 5 domains of the CFIR and synthesised thematically. RESULTS: From 495 articles, a total of 13 eligible studies were identified, with 6 studies using a mixed methods approach, and 7 reporting qualitative methods only. There were 82 implementation factors identified, including 37 barriers and 45 facilitators, and a further 26 recommendations from the papers across all 5 domains of the CFIR. More barriers than facilitators were identified within the CFIR domain inner setting, in contrast to all other domains where facilitator numbers outweighed barriers. CONCLUSIONS: This review identified many facilitators and barriers of implementing physical activity interventions in the community. A key finding of this review was the impact of implementation strategies on successful implementation of community PA interventions. From the evidence, it was clear that many barriers to implementation could have been negated or reduced by an implementation plan in which several strategies are embedded. The findings of this review also suggest more attention to individual' skills and involvement is needed to improve self-efficacy and knowledge. The role of individuals across all organisational levels, from providers to leaders, can impact on the implementation of an intervention and its success. TRIAL REGISTRATION: PROSPERO - CRD42020153821 .

Economic evaluation of 'Men on the Move', a 'real world' community-based physical activity programme for men.

BACKGROUND: Physical activity (PA) interventions capable of producing health benefits cost effectively are a public health priority across the Western world. 'Men on the Move' (MOM), a community-based PA intervention for men, demonstrated significant health benefits up to 52-weeks (W) post-baseline. This article details the economic evaluation of MOM with a view to determining its cost-effectiveness as a public health intervention to be rolled out nationally in Ireland. METHODS: Cost-effectiveness was determined by comparing the costs (direct and indirect) of the programme to its benefits, which were captured as the impact on quality-adjusted life-years (QALYs). For the benefits, cost-utility analysis was conducted by retrospectively adapting various health-related measures of participants to generate health states using Brazier et al.'s (2002) short form-6D algorithm. This in turn allowed for 'utility measures' to be generated, from which QALYs were derived. RESULTS: Findings show MOM to be cost-effective in supporting an 'at risk' cohort of men achieves significant improvements in aerobic fitness, weight loss and waist reduction. The total cost per participant (€125.82 for each of the 501 intervention participants), the QALYs gained (11.98 post-12-W intervention, or 5.3% health improvement per participant) and estimated QALYs ratio costs of €3723 represents a cost-effective improvement when compared to known QALY guidelines. CONCLUSIONS: The analysis shows that the cost per QALY achieved by MOM is significantly less than the existing benchmarks of £20 000 and €45 000 in the UK and Ireland respectively, demonstrating MOM to be cost-effective.

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response.

Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.

Recruiting men from across the socioeconomic spectrum via GP registers and community outreach to a weight management feasibility randomised controlled trial.

BACKGROUND: Men, particularly those living in disadvantaged areas, are less likely to participate in weight management programmes than women despite similar levels of excess weight. Little is known about how best to recruit men to weight management interventions. This paper describes patient and public involvement in pre-trial decisions relevant to recruitment and aims to report on recruitment to the subsequent men-only weight management feasibility trial, including the: i) acceptability and feasibility of recruitment; and ii) baseline sample characteristics by recruitment strategy. METHODS: Men with BMI ≥30 kg/m2 and/or waist circumference ≥ 40 in. were recruited to the feasibility trial via two strategies; community outreach (venue information stands and word of mouth) and GP letters, targeting disadvantaged areas. Recruitment activities (e.g. letters sent, researcher venue hours) were recorded systematically, and baseline characteristics questionnaire data collated. Qualitative interviews (n = 50) were conducted three months post-recruitment. Analyses and reporting followed a complementary mixed methods approach. RESULTS: 105 men were recruited within four months (community n = 60, GP letter n = 45). Community outreach took 2.3 recruiter hours per participant and GP letters had an opt-in rate of 10.2% (n = 90/879). More men were interested than could be accommodated. Most participants (60%) lived in more disadvantaged areas. Compared to community outreach, men recruited via GP letters were older (mean = 57 vs 48 years); more likely to report an obesity-related co-morbidity (87% vs 44%); and less educated (no formal qualifications, 32% vs 10%, degree educated 11% vs 41%). Recruitment strategies were acceptable, a sensitive approach and trusting relationships with recruiters valued, and the 'catchy' study name drew attention. CONCLUSIONS: Targeted community outreach and GP letters were acceptable strategies that successfully recruited participants to a men-only weight management feasibility trial. Both strategies engaged men from disadvantaged areas, a typically underserved population. Using two recruitment strategies produced samples with different health risk profiles, which could add value to research where either primary or secondary prevention is of interest. Further work is required to examine how these strategies could be implemented and sustained in practice. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03040518 , 2nd February 2017.

Reaching beyond the 'worried well': pre-adoption characteristics of participants in 'Men on the Move', a community-based physical activity programme.

BACKGROUND: Issues surrounding gender and men's health have become an increasing focus of public health globally. Unhealthy lifestyles and lower engagement in health promotion initiatives contributed to lower life expectancy and higher mortality rates among men. This study presents the pre-adoption characteristics of men who registered for 'Men on the Move'-a community-based physical activity (CBPA) programme, to ascertain whether the programme reached its intended target group, i.e. 'at-risk' adult men who did not meet physical activity (PA) guidelines and were likely to have multiple risk factors for cardiovascular disease (CVD). METHODS: Multiple recruitment strategies were adopted to engage the target group and baseline data collection included a range of demographic, self-report and outcome measures. RESULTS: The recruitment strategy succeeded in reaching the target group, with the majority (n = 927) presenting being previously inactive (89.0%), overweight/obese (89.7%) and having multiple CVD risk factors (53.1% ≥ 2 risk factors). However, the strategy was less successful in engaging 'hard-to-reach' groups, with the majority being middle-aged, white, married/cohabiting, educated and employed. CONCLUSIONS: A gender-sensitized, partnership and community outreach recruitment strategy can maximize the reach and recruitment of an 'at-risk' cohort for CBPA initiatives, but more targeted approaches are needed to recruit marginalized groups of men.

The impact of a gender-specific physical activity intervention on the fitness and fatness profile of men in Ireland.

BACKGROUND: Amid increasing concerns about rising obesity rates and unhealthy lifestyle behaviours, physical activity (PA) is seen as a prophylactic to many chronic conditions affecting men. Men respond best to community-based PA programmes, using gender-specific promotional and delivery strategies. 'Men on the Move' (MOM) was developed on this basis and targeted inactive adult men in Ireland. METHODS: Sedentary men (n = 927; age = 50.7 ± 10.9 years; weight = 92.7 ± 16.0 kg; METS = 6.06 ± 2.13) were recruited across eight counties: four 'intervention group' (IG; n = 501) and four 'comparison-in-waiting group' (CG; n = 426). The MOM programme involved structured group exercise twice weekly for 12 weeks (W), along with health-related workshops with the groups maintained up to 52 W. Primary outcome measures [aerobic fitness, bodyweight and waist circumference (WC)] together with self-administered questionnaires were used to gather participant data at baseline, 12, 26 and 52 W. RESULTS: Results show a net positive effect on aerobic fitness, bodyweight and WC, with significant (P < 0.05) net change scores observed in the IG compared to the CG (METS: 12 W = +2.20, 26 W = +1.89, 52 W = +0.92; weight: 12 W = -1.72 kg, 26 W = -1.95 kg, 52 W = -1.89 kg; WC: 12 W = -4.54 cm, 26 W = -2.69 cm, 52 W = -3.16 cm). The corresponding reduction in cardiovascular disease risk is particularly significant in the context of a previously inactive and overweight cohort. The high 'dropout' (42.7% presenting at 52 W), however, is of particular concern, with 'dropouts' having lower levels of aerobic fitness and higher bodyweight/WC at baseline. CONCLUSIONS: Notwithstanding dropout issues, findings address an important gap in public health practice by informing the translational scale-up of a small controllable gender-specific PA intervention, MOM, to a national population-based PA intervention targeting inactive men.

Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.

A process evaluation of a Training of Trainers (TOT) model of men's health training.

This study set out to identify the mediators of diffusion of a Training of Trainers (ToT) programme; focusing on ENGAGE, Ireland's national men's health training programme, we explored the process (planning, implementation and maintenance) of using a ToT model of training to affect change in gender sensitive health and social service provision for men. Our findings indicate that an experiential learning approach in combination with mechanisms for feedback and fostering peer-based support during training and beyond are key strategies that foster individual (Trainer), community (of Trainers) and organizational (Trainer workplaces) level ownership. Moreover, by adapting in response to feedback, ENGAGE was able to remain relevant over a number years and to different cohorts of Trainers. As such, core strategies used by ENGAGE could be used to inform new models of health training elsewhere.

From training to practice: the impact of ENGAGE, Ireland's national men's health training programme.

Ireland's National Men's Health Policy recommended developing training programmes tailored to the needs of those working in health and allied health professionals and ENGAGE was developed to meet that recommendation. This study evaluated the impact of ENGAGE on frontline service providers' self-reported knowledge, skills, capacity and practice up to 5-months post training. Between 2012 and 2015, ENGAGE Trainers (n = 57) delivered 62 1-day training programmes to 810 participants. This study was conducted on a subset of those training days (n = 26) and participants. Quantitative methodologies were used to collect pre (n = 295), post (n = 295) and 5-month post (n = 128) training questionnaire data. Overall, participants were highly satisfied with the training immediately post training (8.60 ± 1.60 out of 10) and at 5-month follow up (8.06 ± 1.43 out of 10). Participants' self-reported level of knowledge, skill and capacity in identifying priorities, engaging men and influencing practice beyond their own organisation increased immediately following training (P < 0.001) and, with the exception of improving capacity to engage men and influencing practice beyond their organisation, these improvements were sustained at 5-month post training (P < 0.001). The vast majority of service providers (93.4%) reported that ENGAGE had impacted their work practice up to 5-month post training. The findings suggest that ENGAGE has succeeded in improving service providers' capacity to engage and work with men; improving gender competency in the delivery of health and health related services may increase the utilisation of such services by men and thereby improve health outcomes for men.

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes.

BACKGROUND: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. METHODS AND RESULTS: Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. CONCLUSIONS: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.

A unique protein phosphatase with kelch-like domains (PPKL) in Plasmodium modulates ookinete differentiation, motility and invasion.

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.

Using an Intersectional Approach to Explore the Lived Mental Health Experiences of Traveller Men Affected by Suicide in Ireland.

Rates of suicide are seven times higher among Traveller men, an Indigenous ethnic minority group in Ireland, compared with non-Traveller men. Several factors are implicated, including racism, social exclusion, discrimination, inadequate accommodation, unemployment, and lower educational attainment. Systemic and cultural barriers inhibit Traveller men from seeking support. This study addresses a gap in the literature by exploring the lived mental health experiences of Traveller men affected by suicide. Semi-structured interviews (n = 13; aged 19-50) were conducted with Traveller men affected by suicide. Interviews were recorded and transcribed verbatim. Thematic content analysis was used to analyze the data, which yielded three broad themes. Theme 1, "key determinants of Traveller men's mental health," describes the impact on Traveller men of issues relating to accommodation/homelessness, education, and unemployment, as well as frequent exposure to prejudice, discrimination, and racism. Theme 2, "contemporary Traveller masculinities," considers how Traveller masculinities were shaped by a patrilineal tradition and by historical/ongoing tensions related to their ethnicity. Theme 3, "navigating support seeking and coping with distress," encapsulates both resistant and proactive approaches used by participants to manage their mental health. The intersection of structural inequalities, internalized racism, Traveller masculinities, and strong historical associations between stigma and mental health/suicide within the Traveller community lies at the heart of the heavy burden of suicide carried by Traveller men. Findings provide a deeper understanding of the sources of distress and pathways to resilience/recovery among Traveller men affected by suicide and can inform the development of more gender- and culturally appropriate suicide prevention interventions.

In silico protein interaction screening uncovers DONSON's role in replication initiation.

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.