Tandem immunoaffinity purification of protein complexes from
            <i>Caenorhabditis elegans</i>

Polanowska, Jolanta; Martin, Julie S.; Fisher, R. A.; Scopa, Tina; Rae, Ian F.; Boulton, Simon J.

doi:10.2144/04365bm05

Cited by 35 publications

(35 citation statements)

References 9 publications

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“…Extracts prepared from N2 (wild-type) and dwIs7 C. elegans strains were incubated with MAb 9E10 (Myc) or 12CA5 (hemagglutinin [HA]) coupled to beads for 30 min at 4°C before being washed three times with lysis buffer as previously described (35). Proteins associated with the beads were subjected to electrophoresis and then Western blotting with antibody to BRC-2 or RAD-51.…”

Section: Methodsmentioning

confidence: 99%

RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

Martin

Winkelmann

Petalcorin

et al. 2005

Molecular and Cellular Biology

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171

269

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The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes. 1 The faithful segregation of homologous chromosomes during meiosis is dependent on the formation of physical connections (chiasmata) that form following the successful reciprocal exchange of DNA molecules resulting from crossover recombination. In concert with sister chromatid cohesion, chiasmata facilitate the proper orientation of homologous chromosomes and their subsequent segregation to opposite poles of the meiotic spindle (53). In most eukaryotes, meiotic recombination is initiated by the action of Spo11, a topoisomerase II-like enzyme that catalyzes the formation of meiosis-specific DNA double-strand breaks (DSBs) (15,18). Once formed, a DSB is processed to generate a resected 3Ј single-stranded DNA (ssDNA) tail that is rapidly bound by replication protein A (RPA) before being displaced by RAD51, the eukaryotic homolog of the bacterial DNA strand exchange protein RecA. Rad51 catalyzes DNA strand exchange between homologous sequences, thus promoting the physical exchange of DNA molecules required for successful crossover recombination.

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Section: Methodsmentioning

confidence: 99%

RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

Martin

Winkelmann

Petalcorin

et al. 2005

Molecular and Cellular Biology

Self Cite

171

269

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“…N2 worms were grown in liquid culture in a 35-liter fermentor essentially as described previously (47), collected by sedimentation, and bleached by hypochlorite treatment. Embryos were collected, washed several times in M9 buffer, and snap-frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

CIF-1, a Shared Subunit of the COP9/Signalosome and Eukaryotic Initiation Factor 3 Complexes, Regulates MEL-26 Levels in the Caenorhabditis elegans Embryo

Luke-Glaser¹,

Roy

Larsen

et al. 2007

Molecular and Cellular Biology

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The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.

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“…AP-MS methods have facilitated the generation of largescale protein complex studies in native conditions in Saccharomyces cerevisiae (5,32,33), C. elegans (34), Escherichia coli (35), Drosophila melanogaster (36), and Homo sapiens (37) and have also been used to determine dynamic changes in complex components upon perturbation (38 -43). When compared with other published data sets, such as MIPS (44) manually curated protein complexes, AP-MS interaction data has an estimated FPR between 10 and 40% (32,33,45,46) and reproducibility of experiments estimated at 60 -85% (32,33,46,47).…”

mentioning

confidence: 99%

Popular Computational Methods to Assess Multiprotein Complexes Derived From Label-Free Affinity Purification and Mass Spectrometry (AP-MS) Experiments

Armean

Lilley

Trotter³

2013

Molecular & Cellular Proteomics

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Advances in sensitivity, resolution, mass accuracy, and throughput have considerably increased the number of protein identifications made via mass spectrometry. Despite these advances, state-of-the-art experimental methods for the study of protein-protein interactions yield more candidate interactions than may be expected biologically owing to biases and limitations in the experimental methodology. In silico methods, which distinguish between true and false interactions, have been developed and applied successfully to reduce the number of false positive results yielded by physical interaction assays. Such methods may be grouped according to: (1) the type of data used: methods based on experiment-specific measurements (e.g., spectral counts or identification scores) versus methods that extract knowledge encoded in external annotations (e.g., public interaction and functional categorisation databases); (2) the type of algorithm applied: the statistical description and estimation of physical protein properties versus predictive supervised machine learning or text-mining algorithms; (3) the type of protein relation evaluated: direct (binary) interaction of two proteins in a cocomplex versus probability of any functional relationship between two proteins (e.g., cooccurrence in a pathway, sub cellular compartment); and (4) initial motivation: elucidation of experimental data by evaluation versus prediction of novel protein-protein interaction, to be experimentally validated a posteriori. This work reviews several popular computational scoring methods and software platforms for protein-protein interactions evaluation according to their methodology, comparative strengths and weaknesses, data representation, accessibility, and availability.

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Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans

Cited by 35 publications

References 9 publications

RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

CIF-1, a Shared Subunit of the COP9/Signalosome and Eukaryotic Initiation Factor 3 Complexes, Regulates MEL-26 Levels in the Caenorhabditis elegans Embryo

Popular Computational Methods to Assess Multiprotein Complexes Derived From Label-Free Affinity Purification and Mass Spectrometry (AP-MS) Experiments

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