Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Vera Gorbunova ( vera.gorbunova@rochester.edu ).

Primary fibroblasts were isolated from under-arm skin and lung tissues as previously described (). Briefly, skin tissues were shaved and cleaned with 70% ethanol. Lung tissues were rinsed with PBS to get rid of excess blood. Tissues were minced and incubated in DMEM/F-12 medium (ThermoFisher, 11320033) with Liberase TM (Sigma, 5401127001) at 37°C on a stirrer for 15-90 min. Tissues were then washed and plated with DMEM/F-12 medium containing 15% fetal bovine serum (GIBCO) and Antibiotic-Antimycotic (GIBCO). When cells are ∼80% confluent, isolated cells were frozen in liquid nitrogen within 2 passages. All subsequent cultures were performed in EMEM (ATCC, 30-2003) supplemented with 15% fetal bovine serum (GIBCO), 100 units/mL penicillin, and 100 μg/mL streptomycin (GIBCO). All primary cells were cultured at 37°C with 5% COand 3% Oexcept naked mole-rat cells, which were cultured at 32°C with 5% COand 3% O

All the UAS-Sirt6 transgenic fly lines were generated by PhiC31 integrase-based transgenesis, integrating into the attP40 landing site (BestGene, CA). The Actin-GeneSwitch-Gal4 flies which expresses a progesterone-inducible GAL4 ubiquitously () were courtesy of Dr. Benoit Biteau from University of Rochester Medical Center. All the flies were maintained on standard fly food at 18°C as lab stocks and at 25°C for experiments.

All experiments were performed according to procedures approved by the University of Rochester Committee on Animal Resources (UCAR). Sources of animals used in this study were as previously described (). C57BL/6 mice were purchased from the Jackson Laboratory. Two mice were caught in New York State. Brown Norway rats, BN/Crl, and Fischer 344 rats, F344/IcoCrl, were purchased from Charles River Laboratories. Outbred Mongolian gerbils, Crl:MON(Tum), and outbred golden hamsters, Crl:LVG(Syr), were purchased from Charles River Laboratories. Capybaras were obtained from Bio Fau Assesoria e Comercio (São Paulo, Brazil). Pacas were from the animal facility at São Paulo State University. Outbred multicolored guinea pigs were purchased from Elm Hill Labs. Chinchillas were purchased from Moulton Chinchilla Ranch. Beavers, deer mice, woodchucks, chipmunks, porcupines, red, and gray squirrels were trapped in New York State. Fox squirrels were trapped in Ohio. Blind mole rats were caught in Upper Galilee Mountains in Israel. Naked mole rats were from the colonies at University of Rochester.

Method Details

Host cell reactivation assay Batista et al., 2009 Batista L.F.

Kaina B.

Meneghini R.

Menck C.F. How DNA lesions are turned into powerful killing structures: insights from UV-induced apoptosis. Host cell reactivation assay was used to measure the repair of UV-induced DNA damage. UV-induced DNA lesions are also able to be converted to DNA DSBs during DNA replication by causing collapsed replication forks (). However, the FFL plasmid lacks a eukaryotic replication origin and cannot replicate in rodent cells, ensuring that our assay is specific to measure NER. One mL of a firefly luciferase (FFL) plasmid, pCMV-Luc, at a concentration of 0.5 μg/μL was aliquoted into 25 μL droplets in a 10 cm plate and irradiated with 400 or 1200 J/m2 of 254nm UV light with lid open. The same batch of UV-treated plasmids was used for testing all species to avoid batch-to-batch variation. 1 × 106 cells were co-transfected with 1 μg of treated pCMV-Luc plasmid and 0.04 μg intact Renilla luciferase plasmid pRL-CMV (Promega). Transfections were done with program U-20 using an Amaxa Nucleofector II machine. 48 h after transfection, cells were lysed and luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega). A ratio between firefly luciferase activity and Renilla luciferase activity was calculated to normalize the data. The percentage of repaired DNA was calculated by the recovery of normalized firefly luciferase activity at each UV dose compared with the untreated control. For each cell line, three technical replicates were performed for each UV dose.

UV sensitivity assay and calculation of LD50 Cells were UV irradiated at confluency. Once becoming confluent, the cells in 6cm plates were washed with phosphate-buffered saline (PBS) and irradiated with 254 nm UV light in 5 mL PBS with the lid open. Each plate of a cell line was exposed to serial doses of 0, 25, 50, 100, 200, 400, 800, 1200, or 2400 J/m2. Cells from multiple species receiving the same dose were treated together to minimize variations. Cells from each species were tested in triplicate at each dose. PBS was removed after irradiation and complete medium was added back. Cells were then cultured for 3 days before cell survival was measured by metabolic conversion of WST-1 (Roche). To calculate LD50, the survival percentage at each dose was calculated as the proportion of WST-1 conversion relative to the untreated control. An exponential regression of the cell survival in the function of UV dose was fitted to each cell line. The regression coefficient (r2) between 0.95 and 0.99 was usually achieved. LD50 was then calculated based on the regression equation of each cell line.

Generation of NHEJ and HR reporter cell lines NHEJ and HR reporter constructs were digested with NheI restriction enzyme and purified with the QIAEX II gel extraction kit (QIAGEN). The same batch of DNA preparation was used for generating all reporter cell lines of rodent species. Young cells (population doubling < 10) were recovered from liquid nitrogen and passaged once before integration of the constructs. 0.5 μg of linearized NHEJ and HR constructs were electroporated into each cell line. Two days after transfection, media was refreshed and G418 was applied for selection of the stable clones. Triplicates of each reporter in each cell line were prepared to obtain an adequate number of stable clones. 40-100 clones were generated from each transfection. Clones from triplicate plates were pooled to get at least 100 clones per reporter per cell line. During this process, skin fibroblasts from paca, woodchuck and fox squirrel and lung fibroblasts from paca, gray squirrel, and porcupine gave rise to senescent clones and were not included in further analysis.

DSB repair assays and FACS analysis Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. In vivo DSB repair assays were performed as previously described (). Briefly, growing cells were co-transfected with 5 μg of plasmid encoding I-SceI endonuclease and 0.025 μg of plasmid encoding DsRed. The same batch of I-SceI and DsRed mixture was used throughout all species to avoid batch-to-batch variation. Two human fibroblast cell lines containing clonally integrated NHEJ and HR reporter constructs used previously () were included for each experiment. DSB repair efficiency of these two reference cell lines is consistent throughout the experiments. To test the effect of SIRT6 on DSB repair, SIRT6 plasmids were co-transfected with I-SceI and DsRed plasmids. Three days after transfection, the numbers of GFP+ and DsRed+ cells were determined by flow cytometry on a FACS Canto machine (BD Biosciences). For each sample, a minimum of 20,000 cells were analyzed. DSB repair efficiency was calculated by dividing the number of GFP+ cells by the number of DsRed+ cells.

Cloning Sirt6 from rodents and mutagenesis Sirt6 genes from 7 species including mouse (NM_181586), rat (NM_001031649), golden hamster (XM_005083238), deer mouse (XM_006978115), chinchilla (XM_013505251), naked mole rat (NM_001310264), and blind mole rat (XM_017799169.1) have been annotated and deposited in GenBank. The coding sequence (CDS) of Sirt6 genes from these 7 species were amplified by RT-PCR using mRNA prepared from young cells (PD < 10) of the corresponding species and sequenced to verify the correct cloning. Sirt6 CDS were then cloned into pEGFP-N1 plasmid in which EGFP was replaced. Sirt6 genes of the other rodent species including gerbil, guinea pig, paca, capybara, porcupine, red squirrel, woodchuck, chipmunk, gray squirrel, and beaver were cloned by rapid amplification of cDNA ends (RACE). Degenerate primers designed based on the conserved regions of SIRT6 were used to amplify the internal sequences. Species-specific primers were then designed based on the internal sequences and used in RACE to amplify both 3′ and 5′ end of the Sirt6 cDNA. Full length Sirt6 CDS were finally amplified and cloned into pEGFP-N1 plasmid in which EGFP was replaced. Mutagenesis of mouse and beaver Sirt6 genes was performed in multiple ways. Fusion protein mutants which incorporate a long sequence or multiple sites of the opposite species were generated by DNA synthesis of the mutated regions which were then swapped back into the wild-type Sirt6 genes using KpnI/NotI for mouse Sirt6 and SbfI/NotI for beaver Sirt6. Single mutants were generated using Quickchange II site directed mutagenesis kit (Agilent) by PCR ( Table S5 ). The sequences were verified by Sanger sequencing.

Immunofluorescence and foci quantification Confluent cells from both short- and long-lived species were treated with 8 Gy of γ-radiation, after which DNA damage foci were stained with γ-H2AX and 53BP1 antibodies and quantified at 1h and 24h. Considering the potential non-specificity of γ-H2AX and 53BP1 antibodies across species, we used co-localized foci as a more reliable indication of DNA damage ( Figure S4 A). Fibroblasts were grown on Lab-Tek II Chamber Slides (ThermoFisher Scientific) until reaching confluency, followed by 8 Gy of γ-irradiation. After treatment, cells were fixed at different time points and processed for immunofluorescence. 5-ethynyl-2′-deoxyuridine (EdU) was added 30 min before fixation and stained using the Click–iT Plus EdU Imaging Kit (ThermoFisher Scientific, C10640) to confirm the cell cycle arrest. EdU staining was performed according to the manufacturer’s recommendations. Briefly, the cells were fixed in 4% formaldehyde in PBS for 15 min at RT, after which, cells were rinsed twice with 3% BSA solution in PBS, permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature, washed with 3% BSA in PBS and then treated with reagents of Click-iT cocktail including Azide-Alexa Fluor 647 triethyl ammonium salt in the dark for 30 min. Following EdU labeling, cells were blocked for 1 h at room temperature in 1% Blocking Reagent (Roche, 11096176001) in PBST. For experiments in different species, cells were incubated with mouse monoclonal anti-γH2AX (Millipore, 05-636, 1:500) and rabbit polyclonal anti-53BP1 antibodies (Abcam, ab172580, 1:500) at +4°C overnight. For SIRT6 overexpression experiments, cells were incubated with mouse monoclonal anti-FLAG (Sigma, F3165, 1:500) and rabbit polyclonal anti-γH2AX antibodies (abcam, ab11174, 1:500) at +4°C overnight. All antibodies were diluted in 0.5% Blocking Reagent solution. After incubation with primary antibodies, cells were washed in PBST three times and incubated with goat anti-rabbit (Alexa Fluor 568) and goat anti-mouse antibodies (Alexa Fluor 488) (ThermoFisher Scientific, 1:400) for 1 h at room temperature. Afterward, slides were mounted in VECTASHIELD Antifade Mounting Medium with DAPI. Images were taken using the Leica TCS SP5 Confocal system. Confocal images were collected with a step size of 0.5 μm covering the depth of the nuclei. Far-red, red, green, and blue fluorescence were acquired sequentially to avoid bleed-through artifacts. Lapytsko et al., 2015 Lapytsko A.

Kollarovic G.

Ivanova L.

Studencka M.

Schaber J. FoCo: a simple and robust quantification algorithm of nuclear foci. To count foci, Z stacks of confocal sections were collected, and maximal projections of the series were obtained. Co-localized γH2AX/53BP1 foci in control cells and 24 h samples after irradiation were counted by eye. The foci in 1 h samples were counted using FoCo (), and parameters were adjusted according to the authors’ recommendations.

CRISPR/Cas9-mediated knockout of SIRT6 CRISPR gRNAs were designed using sgRNA Scorer 2.0 and synthesized at Integrated DNA Technologies. The annealed gRNA1 and gRNA2 ( Table S3 ) were inserted into Bbs1-digested pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene plasmid # 62988) to generate PX459-hSIRT6 gRNA1 and PX459-hSIRT6 gRNA2. The plasmids were sequenced to verify the sequence. Tian et al., 2018 Tian X.

Doerig K.

Park R.

Can Ran Qin A.

Hwang C.

Neary A.

Gilbert M.

Seluanov A.

Gorbunova V. Evolution of telomere maintenance and tumour suppressor mechanisms across mammals. To generate SIRT6 knockout human dermal fibroblasts, primary human dermal fibroblasts were first immortalized with a piggyBac vector expressing hTERT () and selected with 1 mg/mL G418 (ThermoFisher). The immortalized cells were co-transfected with PX459-hSIRT6 gRNA1 and PX459-hSIRT6 gRNA2 plasmids and plated sparsely to form colonies in 2 weeks. Colonies were picked and genomic DNA was extracted to perform genotyping PCR. The sequences of the genotyping primers are as follows: F1: 5′-CCGCATTTTAGAGGTGACTGGAG-3′, R1: 5′-CTAGACCCTGACAGCTCTCCTCC-3′; F2: 5′-GACAAGCTGCCTTTTCCAATCCA-3′, R2: 5′-AGGAGGAAAGCAGATGGATGTGT-3′. If the sequence between gRNA1 and gRNA2 is successfully deleted by CRISPR, PCR with the F1 and R1 primers will produce a 400bp band, whereas PCR using the F2 and R2 primers will not amplify anything. F2 and R2 primers will amplify a 367bp band in WT cells. The successfully edited clones were verified using western blotting and used for subsequent experiments.

Construction of lentiviral overexpression vectors The coding sequences (CDs) for mouse WT, mouse 5mut, beaver WT, and beaver 5mut SIRT6 were amplified by PCR using the following primers: pLJM1-mouse Sirt6 primers ( Table S5 ) for mouse WT and mouse 5mut Sirt6, and pLJM1-beaver Sirt6 primers ( Table S5 ) for beaver WT and beaver 5mut Sirt6. The amplified fragments were digested with Age1 and EcoR1, and then inserted between the Age1 and EcoR1 sites of pLJM1-EGFP (Addgene plasmid # 19319). The sequences were verified by Sanger sequencing.

Construction of lentiviral miRNA vectors To knockdown SIRT6 in beaver fibroblasts, we used lentivirus-delivered shRNA. shRNA sequences were designed using Genetic Perturbation Platform (Broad Institute, USA) and BLOCK-iT RNAi Designer (ThermoFisher Scientific). Each pair of oligos ( Table S4 ) were annealed and inserted between the AgeI and EcoRI sites of pLKO.1 hygro (Addgene plasmid # 24150). The sequences were verified by Sanger sequencing.

Lentiviral production and transduction Lentiviral particles were produced in HEK293T/17 cells (ATCC, CRL-11268) using a Lenti-X Packaging kit (Takara, 631275). To transduce pLJM1-based SIRT6 overexpression lentivirus into SIRT6 knockout human dermal fibroblasts, a serial dilution of each lentivirus was transduced in the presence of 10 μg/mL polybrene. Cells were selected with 1 μg/mL puromycin. Control cells were included to ensure the complete death of un-transduced cells. The expression level of SIRT6 was quantified by western blotting. Cells expressing a similar level of different SIRT6 were used for further experiments. To knockdown Sirt6 in beaver skin fibroblasts, pLKO.1-based shRNA lentiviruses were transduced in the presence of 10 μg/mL polybrene. Three days after transduction, cells were selected with 100 μg/mL hygromycin. Control cells were included to ensure the complete death of un-transduced cells. The knockdown efficiency was tested by western blotting.

Clonogenic assay Franken et al., 2006 Franken N.A.

Rodermond H.M.

Stap J.

Haveman J.

van Bree C. Clonogenic assay of cells in vitro. Bodgi et al., 2016 Bodgi L.

Canet A.

Pujo-Menjouet L.

Lesne A.

Victor J.M.

Foray N. Mathematical models of radiation action on living cells: From the target theory to the modern approaches. A historical and critical review. Clonogenic assay was performed according to a previously published protocol () with minor modifications. Basically, cells received γ-radiation at 0, 1, 2, 3, 4, 6, and 8Gy when they were confluent. Serial dilutions of irradiated cells were plated immediately after irradiation. Cells were incubated until the colonies formed, which takes 2-3 weeks except the naked mole rat cells, which take 6-8 weeks. Colonies were fixed and stained in a solution containing 6.0% glutaraldehyde and 0.5% crystal violet and counted by two investigators. Cell survival after each dose of radiation was expressed as the relative plating efficiencies of the irradiated cells to that of the control cells. To calculate the LD50 and LD75, the linear-quadratic model was used to fit the survival data against the radiation dose (). Analyses were performed using Graphpad Prism software.

Senescence-associated β-galactosidase (SA-β-gal) staining Debacq-Chainiaux et al., 2009 Debacq-Chainiaux F.

Erusalimsky J.D.

Campisi J.

Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Dimri et al., 1995 Dimri G.P.

Lee X.

Basile G.

Acosta M.

Scott G.

Roskelley C.

Medrano E.E.

Linskens M.

Rubelj I.

Pereira-Smith O.

et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. 2 . Plates were incubated at 37°C for 16 h without CO 2 . Colorimetric images were taken from different areas of each plate and quantified by a researcher that was blinded to the treatments. Human dermal fibroblasts expressing similar levels of SIRT6 proteins were γ-irradiated at 0, 5, and 10 Gy. Ten days after radiation, cells were splitted to be ∼40% confluent to avoid false-positive staining due to confluency. SA-β-gal staining was performed two days after splitting according to the Campisi lab’s protocol (). Basically, cells were washed twice with PBS and fixed in a solution containing 2% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min at room temperature. After fixation, cells were immediately washed twice with PBS and stained in a solution containing 1 mg/mL 5-bromo-4-chloro-3-indolyl P3-D-galactoside (X-Gal), 40 mM citric acid/sodium phosphate buffer, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl. Plates were incubated at 37°C for 16 h without CO. Colorimetric images were taken from different areas of each plate and quantified by a researcher that was blinded to the treatments.

Protein purification The ORFs of mouse WT, mouse 5mut, beaver WT and beaver 5mut SIRT6 were cloned into the backbone of pET11a vector (Novagen) using NdeI and BamHI ( Table S5 ). Expression vectors were transformed into Rosetta-gami B(DE3)pLysS competent cells (Novagen, 71137-4) and plated on LB agar with ampicillin, kanamycin, tetracycline, and chloramphenicol (AKTC) antibiotics. Single colonies were inoculated in LB medium supplemented with AKTC, and the overnight culture was expanded into 2L AKTC LB medium. When the OD600 reached 0.6-0.8, 1 mM IPTG was added to induce protein production for 3 h at 37°C. The 6 × His-tagged proteins were purified with Ni-NTA beads (ThermoFisher Scientific, R90115).

Histone deacetylation assays Gil et al., 2013 Gil R.

Barth S.

Kanfi Y.

Cohen H.Y. SIRT6 exhibits nucleosome-dependent deacetylase activity. Michishita et al., 2008 Michishita E.

McCord R.A.

Berber E.

Kioi M.

Padilla-Nash H.

Damian M.

Cheung P.

Kusumoto R.

Kawahara T.L.

Barrett J.C.

et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. 2 , 10 μM ZnCl 2 , and 1 mM DTT) for 3 h at 30c°C. After incubation, Laemmli buffer was added, and samples were boiled for 10 min. Histone acetylation levels after reactions were detected using western blot analysis with acetylation specific antibodies. In vitro deacetylation reactions were performed as described in (), with some modifications. 2.5 μM (∼5 μg) of each recombinant SIRT6 protein was incubated with 2 μg purified HeLa polynucleosomes (EpiCypher, 16-0003) in 50 μL deacetylation buffer (50 mM Tris-HCl, pH 8.0, 2mM NAD+, 150 mM NaCl, 4 mM MgCl, 10 μM ZnCl, and 1 mM DTT) for 3 h at 30c°C. After incubation, Laemmli buffer was added, and samples were boiled for 10 min. Histone acetylation levels after reactions were detected using western blot analysis with acetylation specific antibodies.

ADP-ribosylation assay Liszt et al., 2005 Liszt G.

Ford E.

Kurtev M.

Guarente L. Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. 2 , 10 mM DTT, 25 μM unlabeled NAD+ and 8 μCi 32P NAD+ (PerkinElmer, NEG023X250UC) were incubated at 37c°C for 3 h. Samples were then resuspended in Laemmli buffer, and boiled for 10 min. The samples were resolved in SDS-PAGE gels and transferred to polyvinylidene difluoride membrane before autoradiography. The reactions analyzing mono-ADP-ribosylation activity were performed as described previously (), with minor modifications. Reactions containing 2.5 μM (∼5 μg) of each recombinant SIRT6 protein in 50 μL of 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10 μM ZnCl, 10 mM DTT, 25 μM unlabeled NAD+ and 8 μCiP NAD+ (PerkinElmer, NEG023X250UC) were incubated at 37c°C for 3 h. Samples were then resuspended in Laemmli buffer, and boiled for 10 min. The samples were resolved in SDS-PAGE gels and transferred to polyvinylidene difluoride membrane before autoradiography. Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. 2 , 10 μM ZnCl 2 , 1 mM DTT, 400 μM NAD+, and 0.1 μg/mL sonicated fish sperm DNA) at 30c°C for 30 min. PARP1 activity was detected by western blotting with anti-PADPR antibody. The stimulation of PARP1 activity by mouse WT SIRT6, beaver WT SIRT6, and their mutants was performed as in (). Briefly, 2.5 μM (∼5 μg) of recombinant SIRT6 proteins were incubated with 1 μg PARP1 (Sigma, P0996) in 50 μL of PARP1 reaction buffer (50 mM Tris-Cl, pH8.0, 150 mM NaCl, 4 mM MgCl, 10 μM ZnCl, 1 mM DTT, 400 μM NAD+, and 0.1 μg/mL sonicated fish sperm DNA) at 30c°C for 30 min. PARP1 activity was detected by western blotting with anti-PADPR antibody.

Thermal denaturation assay m ) of the mouse WT, mouse 5mut, beaver WT, and beaver 5mut variants of SIRT6. Purified proteins were diluted to 10 μM in a buffer (150 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol) containing 2.5 × SYPRO orange dye (ThermoFisher, S6650). Samples were then aliquoted into 50 μl reaction wells and analyzed in a Bio-Rad CFX Connect Real-Time PCR Detection System. Samples were heated from 10°C to 95°C at a gradient of 0.5°C/min. Fluorescence intensities were measured using the FRET channel and measurements were taken every 0.5°C. At least three replicates were performed for each SIRT6 variant. Each thermal denaturation curve was then normalized so that maximum fluorescence was equal to 1 and minimum fluorescence was equal to 0. The curves were then fitted to the following equation, as previously used in ( Kugel et al., 2015 Kugel S.

Feldman J.L.

Klein M.A.

Silberman D.M.

Sebastián C.

Mermel C.

Dobersch S.

Clark A.R.

Getz G.

Denu J.M.

Mostoslavsky R. Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer. m . I = ( 1 1 + e ( T m − T C ) )

Where I is the normalized fluorescence intensity at temperature T and C is a slope factor. Thermal Denaturation assays were used to determine the melting temperature (T) of the mouse WT, mouse 5mut, beaver WT, and beaver 5mut variants of SIRT6. Purified proteins were diluted to 10 μM in a buffer (150 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol) containing 2.5 × SYPRO orange dye (ThermoFisher, S6650). Samples were then aliquoted into 50 μl reaction wells and analyzed in a Bio-Rad CFX Connect Real-Time PCR Detection System. Samples were heated from 10°C to 95°C at a gradient of 0.5°C/min. Fluorescence intensities were measured using the FRET channel and measurements were taken every 0.5°C. At least three replicates were performed for each SIRT6 variant. Each thermal denaturation curve was then normalized so that maximum fluorescence was equal to 1 and minimum fluorescence was equal to 0. The curves were then fitted to the following equation, as previously used in (), to obtain the TWhere I is the normalized fluorescence intensity at temperature T and C is a slope factor.

SIRT6 de-myristoylation assay 3 in Schuster et al. ( Schuster et al., 2016 Schuster S.

Roessler C.

Meleshin M.

Zimmermann P.

Simic Z.

Kambach C.

Schiene-Fischer C.

Steegborn C.

Hottiger M.O.

Schutkowski M. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions. Schuster et al., 2016 Schuster S.

Roessler C.

Meleshin M.

Zimmermann P.

Simic Z.

Kambach C.

Schiene-Fischer C.

Steegborn C.

Hottiger M.O.

Schutkowski M. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions. + and MYR peptide were indicated in the figure legend. When NAD+ was varied, MYR peptide was held constant at 90 μM. When MYR peptide was varied, NAD+ was held constant at 500 μM. De-MYR rates were calculated from the increase in relative fluorescence per second for a period of 10 min where the rate was linear. Kaleidagraph software was used to fit de-MYR rates to the Michaelis-Menten equation to obtain K M and V m for either NAD or MYR-peptide. Measurements for the entire group of beaver or mouse alleles were performed together, allowing us to directly compared relative fluorescence unit (rfu) changes directly. Since rfu values are comparable, and we added and an identical amount of each SIRT6 protein to each reaction, we present V m /K M values as a proxy for the more traditional k cat /K M . SIRT6 de-myristoylase (de-MYR) activity was determined using a previously described approach using a myristoylated TNF-α peptide (MYR peptide) identical to peptidein Schuster et al. (). In this assay, de-MYR activity is manifest as in increase in fluorescence over time due to the loss of fluorescence quenching of a fluorophore located at the end of the myristoyl group by a neighboring 3-nitrotyrosine group on the peptide (). The peptide we employed was custom synthesized by Genscript (Piscataway, NJ) and its mass was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All de-MYR measurements were performed for 30 min in 25 μL final volume in Greiner 384 well low volume black plates using a Spark 20 M plate reader (Tecan; Männedorf, Switzerland) with λ Ex = 310 nm and λ Em = 405 nm. In all cases 500nM SIRT6 protein was incubated in 50mM Tris-HCl pH 7.5, 150mM NaCl, 10 mM β-mercaptoethanol, and 5% glycerol. When varied, NADand MYR peptide were indicated in the figure legend. When NADwas varied, MYR peptide was held constant at 90 μM. When MYR peptide was varied, NADwas held constant at 500 μM. De-MYR rates were calculated from the increase in relative fluorescence per second for a period of 10 min where the rate was linear. Kaleidagraph software was used to fit de-MYR rates to the Michaelis-Menten equation to obtain Kand Vfor either NAD or MYR-peptide. Measurements for the entire group of beaver or mouse alleles were performed together, allowing us to directly compared relative fluorescence unit (rfu) changes directly. Since rfu values are comparable, and we added and an identical amount of each SIRT6 protein to each reaction, we present V/Kvalues as a proxy for the more traditional k/K

Pulse-chase assays One day after transfection with SIRT6 expressing vectors, adherent cells in 10 cm dish where washed twice in PBS and incubated for 30 min in pulse-labeling medium (DMEM, methionine/cysteine-free and containing 12% dialyzed FBS) to deplete intracellular pools of methionine and cysteine. After the medium was removed, cells were incubated for 1h in a labeling cocktail: Express Protein Labeling Mix [35S] (PerkinElmer) in pulse-labeling medium prepared just before use by adding 200 μCi of [35S] labeling mix in the medium per dish. After labeling, the cocktail was quickly removed and cells were washed twice in PBS and incubated for various time periods in Eagle’s Minimum Essential Medium (EMEM) (ATCC). At desired time points, cells were harvested and subjected to immunoprecipitation procedure. Briefly, cells were lysed in IP buffer (20 mM HEPES pH8, 0.2 mM EDTA, 5% glycerol, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail) for 10 min on ice, and after sonication and centrifugation at 13000rpm at 4°C, lysates were precleared for 1h with Pierce Protein A Agarose (ThermoFisher Scientific), followed by overnight incubation with 3 μg of anti-Sirt6 antibodies (Abcam, ab62739). Lysates were then incubated for 3h with Protein A Agarose, and after 5 washes with IP buffer, immune complexes were eluted with 2x Laemmli sample buffer (BioRad), heated for 10 min at 95°C and loaded on 4%–20% SDS-PAGE gel. After electrophoresis, gels were dried for 2h in gel-drier and exposed to Carestream Kodak BioMax MS films for 9 days before development.

Protein structure analysis Biasini et al., 2014 Biasini M.

Bienert S.

Waterhouse A.

Arnold K.

Studer G.

Schmidt T.

Kiefer F.

Gallo Cassarino T.

Bertoni M.

Bordoli L.

Schwede T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Pettersen et al., 2004 Pettersen E.F.

Goddard T.D.

Huang C.C.

Couch G.S.

Greenblatt D.M.

Meng E.C.

Ferrin T.E. UCSF Chimera--a visualization system for exploratory research and analysis. Mouse WT SIRT6 and its mutant with the five beaver amino acid substitutions were modeled with the SWISS-MODEL program () based on a human SIRT6 structure (PDB code: 3ZG6 ). The two predicted crystal structures were very similar, which were super-imposed for visualization using the UCSF Chimera ().

In vitro co-immunoprecipitation assay In vitro co-immunoprecipitation experiment was used to measure the binding affinity of purified SIRT6 proteins to nucleosomes. 0.25 μM (∼1 μg) of each recombinant SIRT6 protein was incubated with 0.1 μM (∼2.1 μg) HeLa mononucleosomes in 100 μL nucleosome binding buffer (20 mM HEPES pH 8.0, 80 mM KCl, 0.1 mM ZnCl 2 , 0.1% EDTA, 10% glycerol, 0.1% NP40, 0.5 mM DTT) for 20 min at RT. After incubation, 10 μL of each sample was saved to use as input control, and 400 μL IP buffer (20 mM HEPES pH8.0, 0.2 mM EDTA, 5% glycerol, 150 mM NaCl, 1% NP40) supplemented with protease inhibitor cocktail (Roche, 04693132001) and 0.25 μM (∼4 μg) anti-SIRT6 antibody (Abcam, ab62739) were added to each sample. Following rotating at 4°C overnight, 100 μL protein A/G agarose (ThermoFisher Scientific, 20421) was added and tubes were rotated at 4°C for another 2 h. Beads were then collected by centrifugation for 1 min at 8,000 rpm at 4°C and washed 5 times with IP buffer. Proteins were eluted with 2x Laemmli sample buffer (BioRad) and boiled for 10 min. The supernatant after centrifugation was used for western blotting analysis.