PMC:6172693 / 42259-53218 JSONTXT

{"project":"2_test","denotations":[{"id":"30257210-21870263-20058767","span":{"begin":1148,"end":1152},"obj":"21870263"},{"id":"30257210-24928857-20058768","span":{"begin":1172,"end":1176},"obj":"24928857"},{"id":"30257210-21870263-20058775","span":{"begin":1148,"end":1152},"obj":"21870263"},{"id":"30257210-27067600-20058779","span":{"begin":1200,"end":1204},"obj":"27067600"},{"id":"30257210-29480802-20058780","span":{"begin":10246,"end":10250},"obj":"29480802"},{"id":"30257210-19029910-20058786","span":{"begin":4616,"end":4620},"obj":"19029910"},{"id":"30257210-27723750-20058787","span":{"begin":7467,"end":7471},"obj":"27723750"},{"id":"30257210-28650317-20058790","span":{"begin":783,"end":787},"obj":"28650317"},{"id":"30257210-21870263-20058791","span":{"begin":1148,"end":1152},"obj":"21870263"},{"id":"30257210-24928857-20058792","span":{"begin":1172,"end":1176},"obj":"24928857"},{"id":"30257210-27067600-20058793","span":{"begin":1200,"end":1204},"obj":"27067600"},{"id":"30257210-28650317-20058794","span":{"begin":1222,"end":1226},"obj":"28650317"},{"id":"30257210-22673905-20058795","span":{"begin":1244,"end":1248},"obj":"22673905"},{"id":"30257210-24177535-20058796","span":{"begin":1508,"end":1512},"obj":"24177535"},{"id":"30257210-12372305-20058797","span":{"begin":1528,"end":1532},"obj":"12372305"},{"id":"30257210-23940115-20058798","span":{"begin":1782,"end":1786},"obj":"23940115"},{"id":"30257210-19029910-20058799","span":{"begin":4616,"end":4620},"obj":"19029910"},{"id":"30257210-21254760-20058800","span":{"begin":4681,"end":4685},"obj":"21254760"},{"id":"30257210-27686526-20058801","span":{"begin":4801,"end":4805},"obj":"27686526"},{"id":"30257210-28035797-20058802","span":{"begin":6787,"end":6791},"obj":"28035797"},{"id":"30257210-27723750-20058803","span":{"begin":7467,"end":7471},"obj":"27723750"},{"id":"30257210-29480802-20058804","span":{"begin":10246,"end":10250},"obj":"29480802"}],"text":"STAR★Methods\n\nKey Resources Table\n\nContact for Reagent and Resource Sharing\nFurther information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ivan Ahel (ivan.ahel@path.ox.ac.uk).\n\nExperimental Model and Subject Details\nMost of the experiments in these studies utilized recombinant protein and enzymes, as well as chemically synthesized peptides. For the cell biology experiments we used standard human model cell lines U2OS (ATCC HTB-96; osteosarcoma) and HEK293T (ATCC CRL-3216; embryonic kidney). The cells were grown in DMEM (Sigma-Aldrich) supplemented with 10% FBS (GIBCO) and penicillin-streptomycin (100 U/ml, GIBCO) at 37°C with 5% CO2. The generation of ARH3 KO U2OS cells was previously described (Fontana et al., 2017). Absence of mycoplasma contamination confirmed by MycoAlert Mycoplasma Detection Kit.\n\nMethods Details\n\nIn vitro ADPr assays\nA variety of in vitro ADPr assays were used to measure the ability of enzymes to modify or demodify different substrates.\n\nRecombinant proteins and peptides\nRecombinant proteins are purified as described previously (Langelier et al., 2011, Langelier et al., 2014, Gibbs-Seymour et al., 2016, Fontana et al., 2017, Dunstan et al., 2012). Peptides were purchased from EpiCypher or custom made. Nucleosomes were from EpiCypher.\n\nEnzymatic preparation of the modified histone peptides\nRecombinant Dim-5 (the homolog of human SUV39H1/2) and SIRT2 were purified as previously described (Rack et al., 2014, Zhang et al., 2002). Recombinant p300 was purchased from Enzo Life Sciences. HDAC2 was purchased from Active Motif. For histone phosphorylation reactions we used the activated Aurora B fragment called Baronase, which was a gift from the Barr lab (Nunes Bastos et al., 2013). H3 peptides were purchased from EpiCypher. H3 peptides (either WT or Ser-ADPr modified as described above) were incubated in either; HAT buffer (p300) - 50 mM Tris-HCl pH 8.0, 1mM DTT, 100 μM Acetyl-CoA, 10% glycerol for 30 min at 30°C; phosphorylation buffer (Baronase) - 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM DTT for 60 min at 37°C; methyltransferase buffer (Dim-5,) – 50mM Glycine pH 9.8, 2 mM DTT, 10% glycerol for 20 min at room temperature. Reactions were then analyzed by SDS-PAGE and western blotting (detailed below). HDACi reactions (HDAC2, SIRT2) were performed in reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2, which were subsequently supplemented by PARP1/HPF1, activated DNA and 50 μM NAD+ spiked with 32PNAD+. The modification reaction proceeded at room temperature for 20 min before addition of the PARPi Olaparib at 1 μM. Reactions were then analyzed by SDS-PAGE and autoradiography.\n\nStandard radioactivity-based ADPr assay\nRecombinant proteins or peptides were ADPr by PARP1 in the presence or absence of HPF1 and histone peptides. PARP1 concentration in the assays was 1 μM unless stated otherwise, HPF1 was always equimolar to PARP1, histone peptides were used at 0.5 μg per reaction, and recombinant nucleosomes were at 1 μM. The PARP reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2, activated DNA and 50 μM NAD+ spiked with 32PNAD+. The modification reaction proceeded at room temperature for 20 min before addition of the PARPi Olaparib at 1 μM. Reactions were then analyzed by SDS-PAGE and autoradiography.\n\nInverted polarity native gels for ADPr detection\nThis simple, non-radioactive method allows visualization of both substrates and products of ADPr reactions. ADPr reactions were performed as described above, except with 2 μg histone peptide per reaction, and in the presence of non-radioactive NAD+. Samples were mixed with TBE Sample Buffer and loaded on 20% TBE gels in TBE Running Buffer. Samples were run with inverted polarity at 200 V for 1 hr. Gels were then fixed for 30 min in 10% glutaraldehyde, washed in H20 for 3 × 10 min, then stained with Imperial Protein Stain for 1 hr.\n\nIn vitro ADP-ribosyl glycohydrolase assays\nThe assays were performed as in Fontana et al., 2017. Briefly, H3 peptides were incubated with PARP1 and HPF1, under the conditions described above, and stopped by addition of Olaparib. ARH3 was then added to the reactions for incubation at room temperature for 30 min. Reactions were then analyzed by SDS-PAGE and autoradiography. ARH3 concentration was at 1 μM.\n\nReanalysis of published high-quality proteomics datasets\nFor the reanalysis of published high-quality proteomics datasets, public raw files were analyzed with MaxQuant proteomics suite of algorithms (version 1.5.3.17) (Cox and Mann, 2008), using the integrated search engine Andromeda (Cox et al., 2011).\nData from the published proteomics study of peptides enriched with an ADPr-binding macrodomain (Martello et al., 2016) were searched against the human proteome database (downloaded 09.10.2015 from UniProt) with the following parameters: the maximum allowed mass deviation was set to 4.5 ppm for precursor ions and 20 ppm for fragment ions; the minimum peptide length was set to 6 amino acids and the maximum number of missed cleavages was set to 5 with the maximum charge state 7. Variable modifications included acetylation (Protein N-term), Oxidation (M) and ADPr (DEKRSTCYNQHM). The variable modification ADPr allowed for neutral losses of adenine (m/z 136.0618); adenosine with loss of water (m/z 250.0935); AMP (m/z 348.0704); ADP (m/z 428.0367) and ADP-ribose (m/z 542.0684). FTMS top peaks per 100 Da were set to 20. We employed the annotated mass spectrometry (MS)/MS spectra generated by MaxQuant as the basis for our manual validation of spectra. To consider a peptide as modified on Tyr, we required the presence of fragment ions with either the intact ADP-ribose or phosphoribose (resulting from the loss of AMP) pointing to ADPr on Tyr. Unmodified “native” sequence ions were not considered as evidence for localization since it is impossible to distinguish between an original lack of modification and complete loss of ADPr during fragmentation. Two additional pieces of evidence supporting Tyr modification could also be observed in lower mass regions of these spectra. First, a peak matching the immonium ion of modified Tyr (+ ADPr – AMPloss) could be observed (albeit weakly) in these spectra at 330.0742 Da (+1). The native (unmodified) Tyr immonium ion (136.0762) was also generally very weak (∼5%) in comparison to the immediately neighboring Adenine peak (136.0623) in these spectra, as opposed to those of peptides containing Tyr but with ADPR on serine. The significance of this ratio as support of Tyr modification can only be fully assessed with larger numbers of ETD-verified peptide spectra.\nFor the cellular ADPr characterization with HCD and EThcD study (Bilan et al., 2017), variable modifications included oxidation (M), acetylation (Protein N-term and K) and ADPr (DEKRSTYCMNQHM). For confident identification of ADPr sites, we considered only ETD MS/MS spectra and required a minimum Andromeda score of 100, mass deviation smaller than 3 ppm after MaxQuant recalibration and a localization score above 0.9. In addition, we manually validated all the representative spectra by requiring extensive coverage of the peptide backbone fragment ions. For localization we required the clear presence of multiple high-intensity fragment ions pinpointing the modification site.\nFor the cellular MS analysis of endogenous histones study (Leidecker et al., 2016), variable modifications included oxidation (M), acetylation (Protein N-term and K), methylation (KR), dimethylation (K), trimethylation (K) and ADPr (DEKRSTYCMNQHM). Similarly, we considered only ETD MS/MS spectra and required a minimum Andromeda score of 100, mass deviation smaller than 3 ppm after MaxQuant recalibration and a localization score above 0.9.\n\nWestern blotting\nHuman U2OS (ATCC HTB-96) and HEK293T (ATCC CRL-3216) cells were plated and grown overnight in DMEM (Sigma-Aldrich) supplemented with 10% FBS (GIBCO) and penicillin-streptomycin (100 U/ml, GIBCO). To induce DNA damage, cells were incubated with 2 mM H2O2 (Sigma-Aldrich) in DPBS with calcium and magnesium (GIBCO) for the indicated times. For PARPi, cells were pretreated with 10 μM Olaparib for 1 hr, and Olaparib was also added to the DPBS solution in case of the induction of DNA damage. Cells were lysed with Triton X-100 lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1% Triton X-100) at 4°C. Right before use, the buffer was supplemented with 5 mM MgCl2, protease and phosphatase inhibitors (Roche), 1 μM ADP-HPD and 1 μM Olaparib. Benzonase (Sigma) was added to the cell lysates and incubated for 20 min at 4°C. After centrifugation at 14,000 rpm for 15 min, supernatants were collected. Protein concentrations were analyzed by Bradford Protein Assay (Bio-Rad). Proteins were boiled in NuPAGE LDS sample buffer (Invitrogen), resolved on NuPAGE Novex 4%–12% Bis-Tris gels (Invitrogen), and transferred onto nitrocellulose membranes (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in PBS buffer with 0.05% Tween 20 and 5% non-fat dried milk for 1 hr at room temperature, and incubated overnight with primary antibodies at 4°C, followed by 1-hour incubation with peroxidase-conjugated secondary antibodies at room temperature. Blots were developed using ECL (Invitrogen) and analyzed by exposing to films.\n\nImmunoprecipitation experiments\nFlag-immunoprecipitation followed by western blotting was used to analyze the modification status of the precipitated proteins and their mutant versions. Full-length human histone H3.1 and HPF1 cDNAs were cloned into the pDONR221 vector (Thermo Fisher Scientific). Point mutations were produced in pDONR-H3.1 and pDONR-HPF1 using QuikChange Lightning site-directed mutagenesis kit (Agilent). Mammalian expression constructs expressed H3.1 proteins with the C-terminal 3xFlag tag, and HPF1 proteins with N-terminal Flag tag. Wild-type proteins and their mutant versions were expressed in 293T cells. The cells were plated, cultured overnight, and transfected using Polyfect (QIAGEN) with an empty vector or a plasmid expressing the Flag-tagged protein of interest for 24 hr essentially as described (Palazzo et al., 2018). The cell lysates were obtained the same as for the western blotting. Protein concentrations were analyzed by Bradford Protein Assay (Bio-Rad), normalized, and then, the cell lysates were incubated with anti-Flag M2 agarose-conjugated mouse monoclonal antibody (Sigma-Aldrich) for 1 hr while rotating at 4°C. Beads were washed several times with Triton X-100 lysis buffer and eluted with NuPAGE LDS sample buffer (Invitrogen). The samples were then analyzed by Western Blotting as described above.\n\nQuantification and Statistical Analysis\nThe qualitative gel-based assays were used to visualize the experimental results. Representative gels from at least three independent biological replicates were shown. "}