Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

DNA replication stress is a source of genomic instability. Here we identify ​changed mutation rate 1 (​Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that ​Cmr1—together with ​Mrc1/​Claspin, ​Pph3, the chaperonin containing ​TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to ​Cmr1, its human orthologue ​WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that ​Cmr1/​WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins

The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.</p

Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry

Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes

Deletion of Genes Encoding Arginase Improves Use of "Heavy" Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

<div><p>The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast <i>Schizosaccharomyces pombe</i> is hindered by the fact that under normal conditions, arginine is extensively catabolized <i>in vivo</i>, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes <i>car1+</i> and <i>aru1+</i> prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.</p></div

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Structural Organization of the 19S Proteasome Lid: Insights from MS of Intact Complexes

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex