Cells respond to DNA double-strand breaks (DSBs) by recruiting factors such as the DNAdamage mediator protein MDC1, p53-binding protein 1 (53BP1) and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 via phospho-dependent interactions between the RNF8 forkhead-associated (FHA) domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ATM. We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage suggesting that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.DNA DSBs are highly cytotoxic lesions; and to ensure that they are repaired with minimal impact on genome stability, cells mount a complex DNA-damage response (DDR) that includes the spatial reorganization of DSB repair and signaling proteins into subnuclear structures -ionizing radiation-induced foci (IRIF) -that surround DSB sites (1, 2). Most IRIF formation depends on phosphorylation of the histone variant H2AX (to form γH2AX) by the DNA-PK and ATM protein kinases (3-6). The γH2AX epitope is bound by MDC1 (7-10) that then promotes IRIF formation by other proteins, including 53BP1, Nijmegenbreakage-syndrome protein NBS1 and BRCA1 (11,12). BRCA1 recruitment to IRIF requires its interaction with the ubiquitin-binding protein RAP80 (13)(14)(15)(16) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts identify RNF8 as the prime ubiquitin ligase for poly-ubiquitination at DSB sites, define its functional importance in the DDR, and establish how RNF8 is recruited to sites of DNA damage via interactions with MDC1.MDC1 is phosphorylated in an ATM-dependent manner in response to ionizing radiation (IR) (11,12). Potential ATM target sites (consensus S/T-Q) cluster in the MDC1 Nterminus, the most notable being four adjacent motifs conforming to the consensus TQXF (Figs. 1A and S1). Significantly, antibodies raised against a peptide encoding phospho-T719 ( Fig. S2A) indicated that it is targeted by ATM in vitro (Fig. 1B), and in vivo ( Fig. 1C and S2B). However, in vitro assays with bacterially-expressed MDC1 fragments revealed that T719 was not the only site of ATM modification. Phosphorylation was only abolished when an "AQXF" mutant protein bearing threonine-to-alanine substitutions in all four TQXF motifs was used as substrate (Fig. 1D). These data and the recent identification of another TQXF site (T752) as an ATM target (17) therefore imply that MDC1 TQXF motifs are likely all modified by ATM and may function redundantly with one another.To address the function of the MDC1 TQXF motifs, we used siRNA to deplete endogenous MDC1 i...

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem URL.The BACFinder clone search and oligo design tool is available online at http://www.mitocheck.org/cgi-bin/BACfinder.Database accession codes. The ChIP/chip data has been submitted to the Gene Expression Omnibus database with accession number GSE10845. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/naturemethods/. Europe PMC Funders GroupAuthor Manuscript Nat Methods. Author manuscript; available in PMC 2010 May 17. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.At a time when the 'thousand-dollar genome' seems a realistic goal for the near future, methods for dissecting the functions of the encoded genetic information lag far behind the genome sequence, both in throughput and in quality of the produced data. Genome sequencing and subsequent bioinformatics analysis have made it possible to study the function of genes in mammalian tissue culture cells using systematic reverse-genetic approaches1-3 and have radically improved researchers' ability to identify human disease genes. Such studies typically identify single genes, whose biological function has often not yet been described. In order to place the proteins these genes encode in pathways, these studies must be followed by detailed molecular-level analysis, of which the most powerful types are protein localization and protein-protein interaction. The power of protein localization and protein-protein interaction studies can be seen from the genome-wide application of GFP localization and tandem affinity tag-based complex purification in the yeast Saccharomyces cerevisiae, which has produced a comprehensive picture of the core proteome of a simple, well-studied model system4-8. The key advantage of yeast for these studies was their efficient intrinsic homologous recombination, which allowed the same tagcoding sequence to be introduced at the endogenous locus of nearly every gene of the genome. The tagged proteins were then systematically analyzed through standardized, generic, tag-based assays.To transfer this approach to mammali...

Summary Centrioles coordinate the primary microtubule organizing center of the cell and template the formation of cilia, thereby operating at a nexus of critical cellular functions. Here we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of new protein modules in centriole duplication, ciliogenesis and centriolar satellite biogenesis, and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purificationmass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential †

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.

The centrosome is the main microtubule organization centre of animal cells. It is composed of a centriole pair surrounded by pericentriolar material (PCM). Traditionally described as amorphous, the architecture of the PCM is not known, although its intricate mode of assembly alludes to the presence of a functional, hierarchical structure. Here we used subdiffraction imaging to reveal organizational features of the PCM. Interphase PCM components adopt a concentric toroidal distribution of discrete diameter around centrioles. Positional mapping of multiple non-overlapping epitopes revealed that pericentrin (PCNT) is an elongated molecule extending away from the centriole. We find that PCM components occupy separable spatial domains within mitotic PCM that are maintained in the absence of microtubule nucleation complexes and further implicate PCNT and CDK5RAP2 in the organization and assembly of PCM. Globally, this work highlights the role of higher-order PCM organization in the regulation of centrosome assembly and function.