Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks.

Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3' single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.EMBO (long-term fellowship (ALTF 93-2010)), Cancer Research UK (Grant IDs: C6/A11224, C6/A18796, C6946/A14492), La Ligue Nationale Contre le Cancer (senior post-doctoral fellowship, Equipe Labellisée 2013), Wellcome Trust (WT092096), University of Cambridge, INSERMThis is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1288

Chemical inhibition of NAT10 corrects defects of laminopathic cells.

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule "Remodelin" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C-depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.We thank Imagif and Institut de Chimie des Substances Naturelles, centre de recherche CNRS de Gif-sur-Yvette, France, for proteomic analysis. Research in the Jackson laboratory is funded by Cancer Research UK program grant 11 C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDR). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. D.L is funded by an EMBO Long-term fellowship ALTF 834-2011 and by a Project Grant from the Medical Research Council, UK MR/L019116/1, S.B. was funded by an EMBO Long-Term fellowship ALTF 93-2010 and Cancer Research UK. R.R. is supported by the Centre National de la Recherche Scientifique. M.D. is supported by the European Research Council grant DDREAM.

L’inhibition chimique de NAT10 corrige les défauts des cellules laminopathiques

La lamina nucléaire maintient l’architecture du noyau Chez les eucaryotes, la membrane interne du noyau est bordée d’un maillage de protéines fibrillaires appelé lamina nucléaire, composé des protéines dénommées lamines, incluant les lamines A et C [1]. La lamina nucléaire est un élément crucial du maintien de l’architecture et de la forme du noyau, ainsi que de l’organisation globale de la chromatine, car elle sert de plateforme d’ancrage pour l’hétérochromatine et pour les régions télomériques [2, 3]. La lamina fait aussi le lien entre le noyau et le cytosquelette car elle interagit avec des protéines transmembranaires telles que SUN1 (Sad1 and UNC84 domain containing 1) ou les protéines nesprines [4]. Ces propriétés de la lamina expliquent son rôle essentiel dans l’organisation structurelle de la cellule. De ce fait, les mutations des gènes codant pour les lamines sont associées à un large éventail de maladies, regroupées sous le nom de laminopathies [5], dont fait partie le syndrome de vieillissement prématuré Hutchinson-Gilford progeria syndrome (HGPS)

Sampler – open-source data acquisition module for quantum physics

The Sinara hardware platform is a modular, open-source measurement and control system dedicated to quantum applications that require hard real-time performance. The hardware is controlled and managed by the ARTIQ, open-source software that provides nanosecond timing resolution and sub-microsecond latency. The Sampler is a general-purpose precision ADC sampling unit with programmable gain and configurable interface. It is used in numerous applications like laser frequency and intensity servo. This paper presents the Sampler module construction and obtained characteristics

Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites.

The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.We thank Thimo Kurz (University of Dundee, UK) for providing MLN4924 and Kate Dry, Rimma Berlotserkovskaya (S.P.J.’s laboratory), and Eric Lightcap (Takeda Pharmaceuticals) for critical reading of the manuscript. We thank Sylvie Urbe and Michael Clague (University of Liverpool, UK) for providing the GFP-CSN5 plasmid, the Division of Signal Transduction Therapy (University of Dundee, UK) for providing UBE2M and UBE2F plasmids, Matthew Petroski (Sanford-Burnham Medical Research Institute, US) for providing FLAG-UBA3 wild-type (WT) and FLAG-UBA3-A171T constructs, and Nico Dantuma (Karolinska Institute, Sweden) and Changshun Shao (Rutgers University) for providing CUL4A and CUL4B plasmids, respectively. We also thank Nicola Lawrence, Alex Sossick, and Richard Butler (Gurdon Institute, Cambridge, UK) for help with microscopy, Volocity, and Fiji. Research in the S.P.J.’s laboratory is funded by Cancer Research UK programme grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. N.L. is funded by CRUK programme grant C6/A11224, J.S.B. is funded by a Wellcome Trust Clinical Fellowship (WT083416), and Y.G. and M.S.-C. are funded by European Research Council grant DDREAM. S.B. was funded by an EMBO long-term fellowship ALTF 93-2010, Cancer Research UK, and a post-doctoral grant from Ligue Nationale Contre le Cancer. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S2211124715003496

Shear-banding in a lyotropic lamellar phase, Part 2: Temporal fluctuations

We analyze the temporal fluctuations of the flow field associated to a shear-induced transition in a lyotropic lamellar phase: the layering transition of the onion texture. In the first part of this work [Salmon et al., submitted to Phys. Rev. E], we have evidenced banded flows at the onset of this shear-induced transition which are well accounted for by the classical picture of shear-banding. In the present paper, we focus on the temporal fluctuations of the flow field recorded in the coexistence domain. These striking dynamics are very slow (100--1000s) and cannot be due to external mechanical noise. Using velocimetry coupled to structural measurements, we show that these fluctuations are due to a motion of the interface separating the two differently sheared bands. Such a motion seems to be governed by the fluctuations of $\sigma^\star$, the local stress at the interface between the two bands. Our results thus provide more evidence for the relevance of the classical mechanical approach of shear-banding even if the mechanism leading to the fluctuations of $\sigma^\star$ remains unclear

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.publishedVersio

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations