Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.Wellcome Trust [100401/Z/12/Z to M.D.]; EuropeanResearch Council [681299 to F.M.-H.]; Spanish Min-istry of Economy and Competitiveness [BFU2017-83794-PAEI/FEDER, UE to F.M.-H.]; Comunidad de MadridTec4Bio [S2018/NMT-4443 to F.M.-H.]; NanoBioCancer[Y2018/BIO-4747 to F.M.-H.]. Funding for open accesscharge: Wellcome Trust [100401/Z/12/Z].Peer reviewe

Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.[Significance] Single-stranded DNA (ssDNA) is a key intermediate in many cellular DNA transactions, including DNA replication, repair, and recombination. Nascent ssDNA is rapidly bound by the Replication Protein A (RPA) complex, forming a nucleoprotein filament that both stabilizes ssDNA and mediates downstream processing events. Paradoxically, however, the very high affinity of RPA for ssDNA may block the recruitment of further factors. In this work, we show that RPA–ssDNA nucleoprotein filaments are specifically targeted by the human HELB helicase. Recruitment of HELB by RPA–ssDNA activates HELB translocation activity, leading to processive removal of upstream RPA complexes. This RPA clearance activity may underpin the diverse roles of HELB in replication and recombination.Work in the laboratory of M.S.D. was supported by an Elizabeth Blackwell Early Career Fellowship from the University of Bristol (to O.J.W.) and Wellcome Trust Investigator Grant 100401/Z/12/Z (to M.S.D.). Work in the laboratory of E.A. was supported by NIH Grants GM130746 (to E.A.) and GM133967 (to E.A.). F.M.-H. acknowledges support from the European Research Council under European Union Horizon 2020 Research and Innovation Program Grant Agreement 681299. Work in the laboratory of F.M.-H. was also supported by Spanish Ministry of Science and Innovation Grants BFU2017-83794-P (AEI/FEDER, UE; to F.M.-H.) and PID2020-112998GB-100 (AEI/10.13039/501100011033; to F.M.-H.) and Comunidad de Madrid Grants Tec4-Bio–S2018/NMT-4443 (to F.M.-H.) and NanoBioCancer–Y2018/BIO-4747 (to F.M.-H.)

Purified Smc5/6 complex exhibits DNA substrate recognition and compaction

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.The work in the Aragon laboratory was supported by a Wellcome Trust Senior Investigator award to L.A. (100955, “Functional dissection of mitotic chromatin”) and the London Institute of Medical Research (LMS), which receives its core funding from the UK Medical Research Council (MC-A652-5PY00). F.M.-H. acknowledges support from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 681299). Work in the Moreno-Herrero laboratory was also supported by Spanish Ministry of Economy and Competitiveness grant BFU2017-83794-P (AEI/FEDER, UE to F.M.-H.) and Comunidad de Madrid grants Tec4Bio – S2018/NMT-4443 and NanoBioCancer – Y2018/BIO-4747 (to F.M.-H.). Work in the J.T.-R. lab was supported by grants BFU2015-71308-P and PGC2018-097796-B-I00 from the Ministerio de Ciencia, Innovación y Universidades and grant 2017-SGR-569 from AGAUR-Generalitat de Catalunya. The IRBLLEIDA Institute is part of the CERCA Programme-Generalitat de Catalunya

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.Fil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Vranych, Cecilia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Evolution after Anti-TNF Discontinuation in Patients with Inflammatory Bowel Disease: A Multicenter Long-Term Follow-Up Study

OBJECTIVES:The aims of this study were to assess the risk of relapse after discontinuation of anti-tumor necrosis factor (anti-TNF) drugs in patients with inflammatory bowel disease (IBD), to identify the factors associated with relapse, and to evaluate the overcome after retreatment with the same anti-TNF in those who relapsed.METHODS:This was a retrospective, observational, multicenter study. IBD patients who had been treated with anti-TNFs and in whom these drugs were discontinued after clinical remission was achieved were included.RESULTS:A total of 1, 055 patients were included. The incidence rate of relapse was 19% and 17% per patient-year in Crohn''s disease and ulcerative colitis patients, respectively. In both Crohn''s disease and ulcerative colitis patients in deep remission, the incidence rate of relapse was 19% per patient-year. The treatment with adalimumab vs. infliximab (hazard ratio (HR)=1.29; 95% confidence interval (CI)=1.01-1.66), elective discontinuation of anti-TNFs (HR=1.90; 95% CI=1.07-3.37) or discontinuation because of adverse events (HR=2.33; 95% CI=1.27-2.02) vs. a top-down strategy, colonic localization (HR=1.51; 95% CI=1.13-2.02) vs. ileal, and stricturing behavior (HR=1.5; 95% CI=1.09-2.05) vs. inflammatory were associated with a higher risk of relapse in Crohn''s disease patients, whereas treatment with immunomodulators after discontinuation (HR=0.67; 95% CI=0.51-0.87) and age (HR=0.98; 95% CI=0.97-0.99) were protective factors. None of the factors were predictive in ulcerative colitis patients. Retreatment of relapse with the same anti-TNF was effective (80% responded) and safe.CONCLUSIONS:The incidence rate of inflammatory bowel disease relapse after anti-TNF discontinuation is relevant. Some predictive factors of relapse after anti-TNF withdrawal have been identified. Retreatment with the same anti-TNF drug was effective and safe

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)