The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

International audienceDNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated

Nucleosome Chiral Transition under Positive Torsional Stress in Single Chromatin Fibers

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns, at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the (H3-H4)2 tetramer bound with ~50 bp of DNA) obtained by depletion of H2A-H2B dimers, suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented righthanded tetrasome. In view of its low energy, <8 kT, we propose this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer which in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.Comment: 33 pages (double spacing), 7 figure

Octamer displacement and redistribution in transcription of single nucleosomes.

Single nucleosomes were assembled on a 357bp DNA fragment containing a 5S RNA gene from sea urchin and a promoter for SP6 RNA polymerase, and were fractionated as a function of their positions by gel electrophoresis. Transcribed nucleosome positions were detected by observing band disappearance in gels, which in turn provided evidence for the displacement of the histone octamer upon transcription. Differential band disappearance showed that nucleosomes closer to the promoter were harder to transcribe, and transcription was blocked when the nucleosome proximal boundary was at the start site. Nucleosomes located at discrete positions were also eluted from the gel bands and transcribed. In this case, new bands appeared as a consequence of octamer redistribution. Such redistribution occurred over all untranscribed positions, as well as over transcribed positions close enough to the promoter. Similar conclusions were derived from another previously investigated fragment containing a Xenopus 5S RNA gene

Inherited Defects of the ASC-1 Complex in Congenital Neuromuscular Diseases

International audienceDefects in transcriptional and cell cycle regulation have emerged as novel pathophysiological mechanisms in congenital neuromuscular disease with the recent identification of mutations in the TRIP4 and ASCC1 genes, encoding, respectively, ASC-1 and ASCC1, two subunits of the ASC-1 (Activating Signal Cointegrator-1) complex. This complex is a poorly known transcriptional coregulator involved in transcriptional, post-transcriptional or translational activities. Inherited defects in components of the ASC-1 complex have been associated with several autosomal recessive phenotypes, including severe and mild forms of striated muscle disease (congenital myopathy with or without myocardial involvement), but also cases diagnosed of motor neuron disease (spinal muscular atrophy). Additionally, antenatal bone fractures were present in the reported patients with ASCC1 mutations. Functional studies revealed that the ASC-1 subunit is a novel regulator of cell cycle, proliferation and growth in muscle and non-muscular cells. In this review, we summarize and discuss the available data on the clinical and histopathological phenotypes associated with inherited defects of the ASC-1 complex proteins, the known genotype–phenotype correlations, the ASC-1 pathophysiological role, the puzzling question of motoneuron versus primary muscle involvement and potential future research avenues, illustrating the study of rare monogenic disorders as an interesting model paradigm to understand major physiological processes

Human Mismatch Repair Protein MSH6 Contains a PWWP Domain That Targets Double Stranded DNA

International audienceThe eukaryotic mismatch repair (MMR) protein MSH6 exhibits a core region structurally and functionally similar to bacterial MutS. However, it possesses an additional N-terminal region (NTR), comprising a PCNA binding motif, a large region of unknown function and a nonspecific DNA binding fragment. Yeast NTR was recently described as an extended tether between PCNA and the core of MSH6 (1). In contrast, we show that human NTR presents a globular PWWP domain in the region of unknown function. We demonstrate that this PWWP domain binds double-stranded DNA, without any preference for mismatches or nicks, whereas its apparent affinity for single-stranded DNA is about 20 times lower. The S144I mutation, which in human MSH6 causes inherited somatic defects in MMR resulting in increased development of hereditary non polyposis colorectal cancer (2), is located in the DNA binding surface of the PWWP domain. However, it only moderately affects domain stability, and it does not perturb DNA binding in Vitro

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain

is an integral protein of the inner nuclear membrane, which interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2 and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1 / R-Smads interaction, thus perturbing transforming growth factor superfamily signaling pathway. As a first step to understand the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences