Structure of the fanconi anaemia monoubiquitin ligase complex

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair

Purified Smc5/6 complex exhibits DNA substrate recognition and compaction

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.The work in the Aragon laboratory was supported by a Wellcome Trust Senior Investigator award to L.A. (100955, “Functional dissection of mitotic chromatin”) and the London Institute of Medical Research (LMS), which receives its core funding from the UK Medical Research Council (MC-A652-5PY00). F.M.-H. acknowledges support from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 681299). Work in the Moreno-Herrero laboratory was also supported by Spanish Ministry of Economy and Competitiveness grant BFU2017-83794-P (AEI/FEDER, UE to F.M.-H.) and Comunidad de Madrid grants Tec4Bio – S2018/NMT-4443 and NanoBioCancer – Y2018/BIO-4747 (to F.M.-H.). Work in the J.T.-R. lab was supported by grants BFU2015-71308-P and PGC2018-097796-B-I00 from the Ministerio de Ciencia, Innovación y Universidades and grant 2017-SGR-569 from AGAUR-Generalitat de Catalunya. The IRBLLEIDA Institute is part of the CERCA Programme-Generalitat de Catalunya

Whose responsibility is adolescent's mental health in the UK? The perspectives of key stakeholders

The mental health of adolescents is a salient contemporary issue attracting the attention of policy makers in the UK and other countries. It is important that the roles and responsibilities of agencies are clearly established, particularly those positioned at the forefront of implementing change. Arguably, this will be more efective if those agencies are actively engaged in the development of relevant policy. An exploratory study was conducted with 10 focus groups including 54 adolescents, 8 mental health practitioners and 16 educational professionals. Thematic analysis revealed four themes: (1) mental health promotion and prevention is not perceived to be a primary role of a teacher; (2) teachers have limited skills to manage complex mental health difculties; (3) adolescents rely on teachers for mental health support and education about mental health; and (4) the responsibility of parents for their children’s mental health. The research endorses the perspective that teachers can support and begin to tackle mental well-being in adolescents. However, it also recognises that mental health difculties can be complex, requiring adequate funding and support beyond school. Without this support in place, teachers are vulnerable and can feel unsupported, lacking in skills and resources which in turn may present a threat to their own mental well-being

Evaluation of a web-based intervention to reduce antibiotic prescribing for LRTI in six European countries: quantitative process analysis of the GRACE/INTRO randomised controlled trial.

To reduce the spread of antibiotic resistance, there is a pressing need for worldwide implementation of effective interventions to promote more prudent prescribing of antibiotics for acute LRTI. This study is a process analysis of the GRACE/INTRO trial of a multifactorial intervention that reduced antibiotic prescribing for acute LRTI in six European countries. The aim was to understand how the interventions were implemented and to examine effects of the interventions on general practitioners' (GPs') and patients' attitudes