Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

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The NOMAD experiment at the CERN SPS

The NOMAD experiment is a short base-line search for $\nu_{\mu}\rightarrow \nu_{\tau}$ oscillations in the CERN neutrino beam. The $\nu_{\tau}$'s are searched for through their charged-current interactions followed by the observation of the resulting $\tau^{-}$ through its electronic, muonic or hadronic decays. These decays are recognized using kinematical criteria necessitating the use of a light target which enables the reconstruction of individual particles produced in the neutrino interactions. This paper describes the various components of the NOMAD detector: the target and muon drift chambers, the electromagnetic and hadronic calorimeters, the preshower and transition radiation detectors, and the veto and trigger scintillation counters. The beam and data acquisition system are also described. The quality of the reconstruction of individual particles is demonstrated through the ability of NOMAD to observe K$^0_{\rm s}$'s, $\Lambda^0$'s and $\pi^0$'s. Finally, the observation of $\tau^{-}$ through its electronic decay being one of the most promising channels in the search, the identification of electrons in NOMAD is discussed

Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM helicase

International audienceThe Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2 CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1 BLM helicase. Here we identify Rad3 ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1 BLM. We show that direct phosphorylation of Eme1 by Rad3 ATR is essential for catalytic stimulation of Mus81-Eme1. Chk1-mediated phosphorylation also contributes to the stimulation of Mus81-Eme1 when combined with phosphorylation of Eme1 by Rad3 ATR. Both Rad3 ATR-and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1 BLM and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1Δ cell viability. Our findings unravel an elaborate regulatory network that relies on the poorly structured N-terminal domain of Eme1 and which is essential for the vital functions Mus81-Eme1 fulfills in absence of Rqh1 BLM

The SLX4 Complex Is a SUMO E3 Ligase that Impacts on Replication Stress Outcome and Genome Stability

International audienceThe SLX4 Fanconi anemia protein is a tumor suppres- sor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF sub- unit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is medi- ated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO- interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA inter- strand crosslink repair functions. Instead, while detri- mental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is crit- ical to prevent mitotic catastrophe following common fragile site expression

WRNIP1 Protects Reversed DNA Replication Forks from SLX4-Dependent Nucleolytic Cleavage

During DNA replication stress, stalled replication forks need to be stabilized to prevent fork collapse and genome instability. The AAA + ATPase WRNIP1 (Werner Helicase Interacting Protein 1) has been implicated in the protection of stalled replication forks from nucleolytic degradation, but the underlying molecular mechanism has remained unclear. Here we show that WRNIP1 exerts its protective function downstream of fork reversal. Unexpectedly though, WRNIP1 is not part of the well-studied BRCA2-dependent branch of fork protection but seems to protect the junction point of reversed replication forks from SLX4-mediated endonucleolytic degradation, possibly by directly binding to reversed replication forks. This function is specific to the shorter, less abundant, and less conserved variant of WRNIP1. Overall, our data suggest that in the absence of BRCA2 and WRNIP1 different DNA substrates are generated at reversed forks but that nascent strand degradation in both cases depends on the activity of exonucleases and structure-specific endonucleases

Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

In most eukaryotes, genes encoding ribosomal RNAs (rDNA) are clustered in long tandem head-to-tail repeats. Studies of Saccharomyces cerevisiae have indicated that rDNA copy number is maintained through recombination events associated with site-specific blockage of replication forks (RFs). Here, we describe two Schizosaccharomyces pombe proteins, homologs of S. cerevisiae Slx1 and Slx4, as subunits of a novel type of endonuclease that maintains rDNA copy number. The Slx1-Slx4–dependent endonuclease introduces single-strand cuts in duplex DNA on the 3′ side of junctions with single-strand DNA. Deletion of Slx1 or Rqh1 RecQ-like DNA helicase provokes rDNA contraction, whereas simultaneous elimination of Slx1-Slx4 endonuclease and Rqh1 is lethal. Slx1 associates with chromatin at two foci characteristic of the two rDNA repeat loci in S. pombe. We propose a model in which the Slx1–Slx4 complex is involved in the control of the expansion and contraction of the rDNA loci by initiating recombination events at stalled RFs

SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance

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RNA Interference Inhibition of Mus81 Reduces Mitotic Recombination in Human Cells

Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3′ flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo