Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mrel1. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mrel1/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.Epigenetic silencing is a term used to describe the heritable transmission of a transcriptionally inactive state. The silent mating type loci HML and HMR and telomeres of the budding yeast Saccharomyces cerevisiae are examples of loci that undergo this type of transcriptional silencing and have served as a paradigm for studying this process.HML and HMR harbor copies of the mating type information genes, ␣ and a, respectively. They are involved in mating type interconversion with the actively transcribed MAT locus. Transcriptional silencing at these loci relies on the existence of cis-acting DNA regulatory elements, termed silencers (E and I), which flank both loci. These elements recruit the DNA binding proteins Rap1, Abf1, and ORC, which then serve to recruit the silent information regulators (Sir) 1, 2, 3, and 4 (11, 13, 33). The widely accepted view of silencing at these loci (and at telomeres) is that histone tails are deacetylated through the action of Sir2, a NAD-dependent histone deacetylase, creating a binding surface on nucleosomes for the binding of Sir3 and Sir4. Multiple rounds of deacetylation lead to the formation of a Sir2/3/4 polymer that spreads on the nucleosomes of the silent region, altering the chromatin and making it unavailable for transcription. The detailed structure of silent chromatin is not known, and exactly how transcription is prevented is a matter of dispute (6, 34).Sir3 is essential for the establishment and maintenance of the silent state at the HM loci and telomeres. Genetic, twohybrid, and biochemical studies have identified interactions of Sir3 with histones H3 and H4, Sir4, Rap1, Abf1, and Sir3 itself (reviewed in references 11, 13, and 33). Interestingly, all these interactions are within the C-terminal two-thirds of the Sir3 protein. Nevertheless, expression of a Sir3 construct lacking the N-terminal region (hereafter referred to as the N terminus) is not suffici...

The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.

Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 29-PO 4 2À at the splice junction, which is subsequently removed by an essential 29-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 29-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNATyr is fully functional in the Trpt1À/À mouse cells. These observations argue against the presence of a 29-PO 4 2À at the splice junction of ligated RNA molecules in Trpt1À/À cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.

The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromoadjacent homology (BAH) domain found in various chromatinassociated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-Å cocrystal structure of the N-terminal domain of Orc1p in complex with the Orc1p-interacting domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting domain has a bilobal structure: an ␣͞␤ N-terminal lobe and a C-terminal lobe resembling the Tudor domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subdomain and the BAH domain of Orc1p. The structure provides a mechanistic understanding of Orc1p-Sir1p interaction specificity, as well as insights into protein-protein interactions involving BAH domains in general.structure ͉ transcriptional silencing E pigenetic control of gene expression involves the assembly of higher order chromatin structures. In the budding yeast Saccharomyces cerevisiae, cryptic mating-type genes, HML and HMR, are epigenetically silenced. Genetic and biochemical studies have identified cis-and trans-acting factors required for the establishment and maintenance of transcriptional silencing at the HM loci (1). The silent HM loci are flanked by specific cis-acting DNA sequences called the E and I silencers, which contain two or more binding sites for DNA-binding proteins, the origin recognition complex (Orc), Rap1p, and Abf1p (2-6). Silencing at the HM loci also requires four silent information regulator (Sir) proteins, Sir1p, Sir2p, Sir3p and Sir4p (7,8).Orc is a six-protein complex important for initiation of DNA replication and transcriptional silencing (2, 9, 10). The Nterminal domain (NTD) of Orc1p, the largest subunit of Orc, is specifically required for transcriptional silencing at the HM loci (11). The NTD of Orc1p is Ϸ220 residues in length, and it shares Ϸ50% amino acid identity with the N-terminal region of Sir3p. The NTD of Orc1p interacts with Sir1p, and this interaction is important for recruiting other SIR proteins to the HM loci as Sir1p also interacts with Sir4p (12). In HM and telomeric silencing, Sir4p forms a complex with Sir2p, and the Sir2p͞Sir4p complex is joined by . Sir2p is a NAD-dependent histone deacetylase (16-18), whereas Sir3p and Sir4p appear to have structural roles. Sir3p and Sir4p can self-associate, interact with Rap1p, and bind to the N-terminal tails of histone H3 and H4 (19, 20). This complex network of protein-protein interactions is responsible for the assembly of repressive higher-order chromatin structures at the HM loci.The NTD of Orc1p contains a bromo-adjacent homology (BAH) domain (21), which is also present in a number of other chromatin-associated p...