1. Cell Biology
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SIRT6 is a DNA double-strand break sensor

  1. Lior Onn
  2. Miguel Portillo
  3. Stefan Ilic
  4. Gal Cleitman
  5. Daniel Stein
  6. Shai Kaluski
  7. Ido Shirat
  8. Zeev Slobodnik
  9. Monica Einav
  10. Fabian Erdel
  11. Barak Akabayov
  12. Debra Toiber  Is a corresponding author
  1. Department of Life Sciences, Ben-Gurion University of the Negev, Israel
  2. The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Israel
  3. Department of Chemistry, Ben-Gurion University of the Negev, Israel
  4. Division of Chromatin Networks, German Cancer Research Center (DKFZ), BioQuant, Germany
  5. Centre de Biologie Intégrative, CNRS UPS, France
Research Article
Cite this article as: eLife 2020;9:e51636 doi: 10.7554/eLife.51636
11 figures, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
SIRT6 arrives at sites of damage independently of other repair factors.

(A–C) Imaging and AUC for SIRT6-GFP in cells with or without Olaparib. (A) Live imaging recruitment upon UV laser-induced damage (LID) shown by SIRT6-GFP in U2OS +/– Olaparib. Representative experiment examining SIRT6 recruitment to LID (n[+Ola]=23, n[–Ola]=23). (B) SIRT6 accumulation in same experiment as panel (A). (C) Average area under the curve (AUC) for cells +/– Olaparib in three replicate experiments. Error bars are the standard error of the mean (SEM) (n[+Ola]=38, n[–Ola]=39, p>0.05). (D–F) Imaging and AUC for SIRT6-GFP accumulation in shControl, shKu80 or shMRE11 Hela cells. (D) Average AUC from three experiments. Error bars are the SEM (shControl: n = 50; shKu80: n = 50, p<0.0005; shMRE11: n = 52, p>0.05). Accumulation of SIRT6-GFP (E) and imaging (F) from a representative experiment examining SIRT6 recruitment after LID (n[shControl]=28; n[shKu80]=30, n[shMRE11]=30). (G–I) MRE11-Cherry accumulation in response to LID in SIRT6 WT and KO U2OS cells. MRE11-Cherry imaging (G) and accumulation (H) in a representative experiment (n[WT]=20, n[KO]=16). (I) Mean AUC for three replicate experiments. Error bars are the SEM (n[WT]=36, n[KO]=33, p<0.0005). (J–L) Ku80-GFP accumulation in response to LID in SIRT6 WT and KO U2OS cells. Ku80-GFP imaging (J) and accumulation (K) in a representative experiment (n[WT]=17, n[KO]=17). (L) Mean AUC for three replicate experiments. Error bars are the SEM (n[WT]=33, n[KO]=33, p>0.05).

Figure 1—figure supplement 1
SIRT6 arrivesatsites of damage independently of other repair factors.

(A, B) Effect of LID on Macro-domain mKate2 in U2OS +/- Olaparib. (n[+Ola]=10, n[-Ola]=10). (C) Average AUC of Macro-mKat2 in U2OS +/- Olaparib for three replicated experiments (error bars are SEM) (n[+Ola]=25, n[–Ola]=25, p<0.0005). (D) Blot of shControl and shMRE11 Hela cells. (E) Effect of LID on NBS1-Cherry in shControl (n = 10) and shMRE11 Hela cells (n = 10). (F) Average AUC for three replicate experiments +/- SEM (n[shControl]=29, n[shMRE11]=30, p<0.005)). (G) Protein blot of shControl and shKu80 Hela cells. (H) Effect of LID on Ku70-GFP accumulation in shControl (n = 10) and shKu80 Hela cells (n = 10). (I) Average AUC for three replicate experiments. Error bars are SEM (n[shControl]=33, n[shKu80]=31, p<0.0001). (J) Blot of WT and SIRT6KO cells.

Figure 1—figure supplement 2
SIRT6 arrivesatsites of damage independently of other repair factors.

(A) Blot of shATM, shH2AX and shcControl Hela cells. (B) Immunofluorescence of γH2AX in sh-Hela cells upon irradiation (IR). (C) Quantification of γH2AX foci in Control (n[–IR]=283, n[+IR]=301), shH2AX (n[–IR]=284, n[+IR]=322) and shATM (n[–IR]=299, n[+IR]=385) Hela cells. The bar chart shows the percentages of cells with 0–3, 4–10 or more than 10 foci per nucleus (+/- IR, average of six experiments). (D) Statistical analysis of the γH2AX foci quantification experiment depicted in panel (C) (*, p < 0.05; **, p <0.005, ***, p < 0.0005). (E,F) Representative experiment showing SIRT6-GFP accumulation in response to LID in shH2AX (n = 10), shATM (n = 10) or shControl (n = 10) cells. (G) Average AUC for three replicate experiments. Error bars are SEM (shControl: n = 30; shH2AX: n = 30, p>0.05; shATM: n = 30, p>0.05).

Figure 2 with 1 supplement
SIRT6 is not recruited by signaling.

(A) Schematic representation of the ‘Tethering assay’. Recruitment can occur through DDR signaling (ATM-LacR-Cherry) or through direct protein–protein interaction (SNF2H-LacR-GFP). (B, C) Recruitment of SIRT6-GFP/SIRT6-Cherry to LacO sites by ATM-LacR-Cherry (n = 30, p>0.05), SNF2H-LacR-GFP (n = 85, p<0.005) and GFP-LacR (n = 85). The bar chart in panel (B) depicts averages for3–6 experiments. Error bars are SEM.

Figure 2—figure supplement 1
SIRT6 is not recruited by signaling.

(A, B) Initiation of DDR by ATM-LacR-Cherry (n = 33, p<0.005), NBS1-LacR-Cherry (n = 82, p<0.05), MRE11-LacR-Cherry (n = 136, p<0.005) and Ku80-LacR-GFP (n = 52, p>0.05) in the tethering system. Co-localization percentage with γH2AX (compared to GFP-LacR (n = 310)) (A) and immunofluorescence (B) are shown. (C, D) Recruitment of SIRT6-GFP/SIRT6-Cherry to LacO sites by NBS1-LacR-Cherry (n = 87, p<0.0005), MRE11-LacR-Cherry (n = 31, p<0.005), Ku80-LacR-GFP (n = 45, p<0.005) and GFP-LacR (n = 85). Co-localization percentage with SIRT6 (compared to GFP-LacR (C) and immunoflorescence (D) are shown. Averages are for 3–5 experiments. Error bars show the SEM.

Figure 3 with 2 supplements
SIRT6 binds DNA with no intermediates.

(A–B) Gel retardation assay of 32 P-5' end-labeled single-strand DNAs and sticky ended dsDNAs as a function of increasing concentrations of SIRT6-His (ssDNA, Kd = 1.48 ± 0.52; sticky dsDNA, Kd = 3.59 ± 0.17). (C) Suggested model of SIRT6 binding to ssDNA as a monomer or open ssDNA ends of dsDNA as a dimer. (D) Ability of SIRT6-Flag to bind to the DNA of circular, blunt-ended and sticky-ended cleaved plasmids. The bar chart depicts averages for three replicate experiments (error bars show SEM), after logarithmic transformation. (E) SIRT6-Flag DNA-binding ability for an open-ended +plasmid +/- NAD. Data are averages from four experiments, with error bars representing SEM (after logarithmic transformation). (F, G) Dimerization of SIRT6 at the LacO site, represented by the recruitment of SIRT6-Cherry by SIRT6-LacR-GFP (n = 181, p<0.005) or GFP-LacR (n = 104). Data are averages from four experiments, with error bars representing SEM.

Figure 3—figure supplement 1
SIRT6 binds DNA with no intermediates.

(A) Gel retardation assay of 32 P-5′ end-labeled dsDNAs with blunt ends as a function of increasing concentrations of SIRT6-His. (B) Gel retardation assay of 32 P-5′ end-labeled RNAs as a function of increasing concentrations of SIRT6-His. Kd could not have been calculated with a 95% confidence interval (CI). (C) DNA-binding ability of SIRT6-Flag, MRE11-Flag and NBS1-Flag for circular and linear plasmids, assessed by a DNA-binding assay. Data are means for 3–9 experiments (with error bars representing SEMs) after logarithmic transformation. (D) SIRT6-Flag DNA-binding ability for plasmids with a 3′ over-hang and a 5′ over-hang assessed by a DNA-binding assay. Log of averages of three experiments (with error bars representing SEMs). (E) Relative amount of NAD+ remaining after consumption by SIRT6 in the presence of DNA alone or with an acetylated peptide (H3K56ac). (F, G) Protection of sticky-ended and blunt-ended plasmids by BSA, SIRT6 and MRE11 against Exonuclease 1 in different time points. Data in panel (G) are means (with error bars showing +/- SEM) from three experiments. (H) Statistical analysis of the end protection: amounts of uncleaved DNA remaining in the presence of SIRT6 or MRE11 compared to BSA (as a control) after 0, 10 or 20 min (*, p <0.05; **, p <0.005; ***, p <0.0005).

Figure 3—figure supplement 2
SIRT6 binds DNA with no intermediates.

(A) SEC-MALS analysis of SIRT6 or of SIRT6 with a dsDNA 10-bp oligo with 3-bp overhanging ends on both sides. For SIRT6: Peak1 protein mass (calculated by UV) = 66.7 ± 3.3 kDa, protein mass (calculated by RI) = 70.7 ± 3.5 kDa; Peak2 protein mass (calculated by UV) = 114.3 ± 5.7 kDa, protein mass (calculated by RI) = 103.8 ± 4.9 kDa; Peak3 protein mass (calculated by UV) = 609.7 ± 14.6 kDa, protein mass (calculated by RI) = 833.6 ± 16.4 kDa. For SIRT6 +DNA: Peak1 protein mass (calculated by UV) = 52.5 ± 3.1 kDa, protein mass (calculated by RI) = 55.7 ± 3.2 kDa; Peak2 protein mass (calculated by UV) = 99.4 ± 3.8 kDa, protein mass (calculated by RI) = 94.6 ± 3.5 kDa; Peak3 protein mass (calculated by UV) = extremely high, protein mass (calculated by RI) = 9224 ± 258 kDa. (B) X-ray scattering profile (right) and the distance distribution function (left) of SIRT6 (blue) and SIRT6 bound to dsDNA (red). (C) Overall parameters for small angle X-ray scattering of SIRT6 alone and of SIRT6 bound to dsDNA determined from the distance distribution function P(r). Rg is the radius of gyration, and Dmax is the maximum dimension of the particle. (D) SAXS structure of SIRT6 (grey surface). Ab initio models were reconstructed from SAXS data using the computer program DAMMIN (Svergun, 1999) and were averaged by the computer program DAMAVER (Volkov and Svergun, 2003). The crystal structure of SIRT6 tetramer (grey spheres) was extracted from the crystal structure of SIRT6 (pdb id: 3PKI) and compared with the obtained SAXS model in PyMOL (http://www.pymol.org).

Figure 4 with 1 supplement
SIRT6 binds DSB through its core domain.

(A) Predicted DNA-binding site based on the published SIRT6 structure, (http://dnabind.szialab.org/). Highlighted in yellow are the predicted DNA-binding amino acids in the SIRT6 core domain; red highlights show the tunnel-forming amino acids that were mutated. (B) Schematic representation of the SIRT6 core domain. (C) List of amino acids that are predicted to participate in the ‘tunnel-like’ structure. (D) DNA binding of an open-ended plasmid by full-length SIRT6 (p<0.0005) and by the SIRT6-core domain (p<0.005). Data are the log of averages from three experiments (with error bars respresenting SEMs). (E) SIRT6 ssDNA-binding prediction, based on the known SIRT6 structure with bound ssDNA. (F, G) Gel retardation assay of 32 P-5′ end-labeled ssDNAs with SIRT6-MBP mutants. Data are averages from three experiments (with error bars representing SEMs). (H) Ability of Flag-tagged mammalian Sirtuins to bind the DNA of circular and linear plasmids. Data are averages from 4–7 experiments (with error bars representing SEMs) (*, p <0.05; **, p <0.005; ***, p <0.0005).

Figure 4—figure supplement 1
SIRT6 binds DSB through itscore domain.

(A) List of generated SIRT6 point mutations in the SIRT6 tunnel structure. (B) Ponceau staining of SIRT6-MBP point mutants. (C) Catalytic activity of SIRT6-MBP mutants, assessed by H3K9 de-myristolation in a FLUOR DE LYS assay. (D) Ability of His-tagged mammalian Sirtuins to bind the DNA circular and linear plasmids. Data are logs of the averages from 4–7 experiments (with error bars representing SEMs) (*, p<0.05; **, p<0.005; ***, p<0.0005).

Figure 5 with 1 supplement
SIRT6 can initiate the DNA damage response.

(A, B) Initiation of the DDR, measured by co-localization of MRE11-LacR-Cherry (n = 136, p<0.005), SIRT6-LacR-GFP (n = 243, p<0.0001) or SIRT6 HY-LacR-GFP (n = 71, p<0.0001), compared to GFP-LacR (n = 310). Data are means for 4–9 experiments (error bars are SEMs). (C, D) Live imaging upon laser-induced damage (LID) of SIRT6-WT-GFP (n = 20) or SIRT6-HY-GFP (n = 20) in SIRT6 KO U2OS cells (n = 20). (D) Accumulation over time in 3 s intervals. (E) Average area under the curve of for three LID experiments (error bars are SEMs) (n[S6-WT]=40, n[S6-HY]=39, p<0.0001). (F) Initiation of DDR by full-length SIRT6-LacR-GFP (n = 127, p<0.005), Core-LacR-GFP (n = 66, p>0.05) or GFP-LacR (n = 64). Data are averages from 3–4 experiments (error bars show SEMs). (G) Initiation of DDR by SIRT6-LacR-GFP (n = 127, p<0.05), SIRT1-LacR-GFP (n = 44, p>0.05), SIRT2-LacR-GFP (n = 67, p<0.005), SIRT7-LacR-GFP (n = 68, p<0.005) or GFP-LacR (n = 64). Data are averages from 3–10 experiments (error bars show SEMs).

Figure 5—figure supplement 1
SIRT6 can initiatetheDNA damage response.

(A) Western blot of nuclear protein fraction of LacO containing U2OS cells after 12 or 24 hr treatment with 0, 5 or 10 mM nicotinamide (NAM) added to their media. (B) Co-localization percentage with γH2AX of SIRT6 HY-LacR-GFP and GFP-LacR after 24 hr of treatment with NAM at three concentrations (*, p<0.05; **, p<0.005; ***, p<0.0005). (C) Immunofluorescence (IF) of co-localization of Core-LacR-GFP (n = 66, p>0.05). Experiment quantification appears in Figure 5C. (D) IF of co-localization of SIRT1-LacR-GFP (n = 44, p>0.05), SIRT2-LacR-GFP (n = 67, p<0.005) and SIRT7-LacR-GFP (n = 68, p<0.005). Experiment quantification appears in Figure 5D.

Figure 6 with 3 supplements
SIRT6 can recruit enzymes of both the NHEJ and HR repair pathways.

(A, B) Percentage repair enzymes that are co-localized with SIRT6-LacR-GFP/Cherry at LacO sites. IF with Flag antibody. Data are averages for 3–6 experiments (with error bars representing SEMs) (*, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005).

Figure 6—figure supplement 1
SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

(A) Laser-induced damage imaged by IF 30 min after damage. The images show recruitment of p-ATM, 53BP1 or BRCA1 in shControl or shSIRT6 U2OS.

Figure 6—figure supplement 2
SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

(A, B) Co-localization of 53BP1 and BRCA1 with SIRT6-LacR-GFP (53BP1: n = 247, p<0.005; BRCA1: n = 223, p<0.005) and HY-LacR-GFP (53BP1: n = 113, p<0.005; BRCA1: n = 122, p<0.005), compared with GFP-LacR (53BP1: n = 130; BRCA1: n = 157). The data shown in panel (B) are averages from 3–5 experiments (error bars are SEMs). (C, D) Comparison of co-localization of endogenous 53BP1 (n = 247) and BRCA1 (n = 223) and transfected 53BP1-Flag (n = 111) and BRCA1-Flag (n = 146) with SIRT6-LacR-GFP, assessed by IF.

Figure 6—figure supplement 3
SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

(A) Schematic representation of the Tethering assay with inhibition of signaling using Wortmannin. (B–D) Co-localization of γH2AX (B), 53BP1 (C), and BRAC1 (D) with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). (B) Co-localization with γH2AX (0 μM: n[SIRT6] = 43, n[GFP] = 41; 1 μM: (n[SIRT6] = 42, n[GFP] = 42; 10 μM: (n[SIRT6] = 42, n[GFP]=41). (C) Co-localization with 53BP1 (0 μM: n[SIRT6] = 32, n[GFP] = 32; 1 μM: n[SIRT6] = 30, n[GFP] = 32; 10 μM: n[SIRT6] = 33, n[GFP] = 31). (D) Co-localization with BRCA1 (0 μM: n[SIRT6] = 30, n[GFP] = 32; 1 μM: n[SIRT6] = 31, n[GFP] = 31; 10 μM: n[SIRT6] = 32, n[GFP] = 31). (E) Co-localization of Ku80-Flag or MRE11-Flag with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). For Ku80-Flag — 0 μM: n[SIRT6] = 45, n[Cherry] = 53; 10 μM: n[SIRT6] = 45, n[Cherry] = 58. For MRE11-Flag — 0 μM: n[SIRT6] = 50, n[Cherry] = 56; 10 μM: n[SIRT6] = 60, n[Cherry] = 51. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005).

Author response image 1
Aspartate basepairing with DNA bases and backbone.
Author response image 2
Scheme of Laser Induced damage (LID) experiment.
Author response image 3
Western blot of Hela cells after attempt to create PARP1 KO using a CRISPR-Cas9 system.
Author response image 4
Co-localization analysis.

(A) LacR-ATM with SIRT6-GFP: Both are nuclear proteins that are usually found evenly spread across the entire nucleus. While we can see a sharp peak where the ATM-LacR focus is located, we do not see a peak of SIRT6-GFP at the same cellular location, which means there is no co-localization of the two proteins in this image. (B) LacR-SNF2H with SIRT6-RFP: A clear peak in the graph of the SNF2H-LacR is accompanied by a clear peak of the SIRT6-RFP, meaning positive co-localization. Since the red signal is much weaker than the green, the SIRT6 peak is much smaller than the SNF2H one. (C) LacR-Ku80 with SIRT6-RFP: In this case the SIRT6-RFP signal is much stronger that the Ku80-LacR GFP signal; however, we can still observe clear peaks at the same location. (D) LacR-GFP with SIRT6-RFP: In this negative control condition, there are no parallel peaks in the graph. However, when looking at the merged image we see that both cells are”yellow”. This just means that both signals are equally as strong (as can be seen in the graph as well) and that both proteins are spread throughout the entire nucleus; however, this is not a co-localization. (E-F) LacR-SIRT6 with 53BP1: When comparing endogenous 53BP1 (E) to the exogenous (F), we can see that using an endogenous antibody usually gives a “cleaner” higher peak with less background; however, in both cases the co-localization is clear.

Author response image 5
IF for ɤH2AX in in SIRT6-LacR-GFP expressing U2OS cells.

“New” vs. “Old” cells.

Tables

Table 1
DNA sequences used in the EMSA assay.
DNA sequences used in the EMSA assays
ssDNA5′GGGAAAGTTGACGGGAGGGTATTGGAGGTTAGTGGAGGTGAGTGG3′
ssDNA5′CCACTCACCTCCACTAACCTCCAATACCCTCCCGTCAACTTTCCC3′
dsDNA-Blunt5′CCACTCACCTCCACTAACCTCCAATACCCTCCCGTCAACTTTCCC3
dsDNA-recessed5′CCACTCACCTCCACTAACCTCCAATACCCTCCCGTCAAC3′
dsDNA-recessed5′ACCTCCACTAACCTCCAATACCCTCCCGTCAACTTTCCC3′
dsDNA-Blunt5′AAGGTCGACACCACCTTTGAGAGCGCGCGGCCCACGCAGACCCACATGGCGCTGGTGCAGCTGGAGCGCGTGGGCCTCCTCCGCTTCCTGGTCAGCCAGAACGTCGACAAA3′
dsDNA-recessed5′TCGACACCACCTTTGAGAGCGCGCGGCCCACGCAGACCCACATGGCGCTGGTGCAGC
TGGAGCGCGTGGGCCTCCTCCGCTTCCTGGTCAGCCAGAACG
3′
RNA5′GCGAAGUCUUCGU3′
Table 2
Theoretical Rg (Å) derived from the SAXS data.
Theoretical Rg (Å)
SIRT6 dimer27.14
SIRT6 tetramer36
SIRT6 hexamer40
Key resources table
Reagent type
(species) or resource
DesignationSource or
reference
IdentifiersAdditional
information
Recombinant DNA reagentATM-LacR-CherrySoutoglou and Misteli, 2008
Recombinant DNA reagentCDT1-TagRFPThermoFisherP36237
Recombinant DNA reagentCMV-FlagToiber et al., 2013
Recombinant DNA reagentMRE11-FlagWu et al., 2008
Recombinant DNA reagentMRE11-LacR-CherrySoutoglou and Misteli, 2008
Recombinant DNA reagentmRFP-SIRT6Kaidi et al., 2010 - retracted
Recombinant DNA reagentNBS1-FlagWu et al., 2008
Recombinant DNA reagentNBS1-LacR-Cherry
Recombinant DNA reagentpcDNA3.1(+)Flag-His-ATM-WTAddgene31985
Recombinant DNA reagentpcDNA5-FRT/T0-Flag-53BP1Addgene52507
Recombinant DNA reagentpDEST 3x Flag-pcDNA5-FRT/T0-BRCA1Addgene52504
Recombinant DNA reagentpEGFP-C1-FLAG-Ku70Addgene46957
Recombinant DNA reagentpEGFP-C1-FLAG-Ku80Addgene46958
Recombinant DNA reagentpEGFP-C1-FLAG-XRCC4Addgene46959
Recombinant DNA reagentpEGFP- SIRT6Kaidi et al., 2010 - retracted
Recombinant DNA reagentpET28 hSIRT6-HisGertman et al., 2018
Recombinant DNA reagentpHPRT-DRGFPAddgene26476
Recombinant DNA reagentpMal-C2-hSIRT6-WTGertman et al., 2018
Recombinant DNA reagentpMal-C2-hSIRT6-A13WThis paper
Recombinant DNA reagentpMal-C2-hSIRT6-D63HThis paper
Recombinant DNA reagentpMal-C2-hSIRT6-D63YThis paper
Recombinant DNA reagentpMal-C2-hSIRT6-H133YGertman et al., 2018
Recombinant DNA reagentpMal-C2-hSIRT6-D188AThis paper
Recombinant DNA reagentpMal-C2-hSIRT6-D190WThis paper
Recombinant DNA reagentpMal-C2-hSIRT6-I217AThis paper
Recombinant DNA reagentpQCXIP-Cherry-LacRThis paper
Recombinant
DNA reagent
pQCXIP-SIRT6 Core-GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-GFP-LacRAddgene59418
Recombinant DNA reagentpQCXIP-KU80-GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-mSIRT6-Cherry-LacRThis paper
Recombinant DNA reagentpQCXIP-mSIRT6-GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-mSIRT6-H133Y-GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-SIRT1-GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-SIRT2- GFP-LacRThis paper
Recombinant DNA reagentpQCXIP-SIRT7- GFP-LacRThis paper
Recombinant DNA reagentSIRT1-FlagZhong et al., 2010
Recombinant DNA reagentSIRT2-FlagAddgene13813
Recombinant DNA reagentSIRT3-FlagAddgene13814
Recombinant DNA reagentSIRT4-FlagAddgene13815
Recombinant DNA reagentSIRT5-FlagAddgene13816
Recombinant DNA reagentSIRT6 CoreTennen et al., 2010
Recombinant DNA reagentmSIRT6-WT-FlagZhong et al., 2010
Recombinant DNA reagentSIRT7-FlagAddgene13818
Recombinant DNA reagentSNF2H-WT-GFP-LacRKlement et al., 2014
AntibodyAlexa Fluor
488 AffiniPure Donkey Anti-Rabbit IgG (H+L)
Jackson Immunoresearch711-545-152IF (1:200)
AntibodyAlexa Fluor
594 AffiniPure
Donkey Anti-Rabbit IgG (H+L)
Jackson Immunoresearch711-585-152IF (1:200)
AntibodyAlexa Fluor
488 AffiniPure Donkey Anti-Mouse IgG (H+L)
Jackson Immunoresearch715-545-150IF (1:200)
AntibodyAlexa Fluor
594 AffiniPure Goat Anti-Mouse IgG (H+L)
Jackson Immunoresearch115-585-062IF (1:200)
Antibody53
BP1
Snata-Cruz Biotechnologysc-22760IF (1:300)
AntibodyBRCA1Snata-Cruz Biotechnologysc-7298WB (1:1000)
AntibodyFlagSigma-AldrichF1804IF (1:900), WB (1:1000)
AntibodyFlag BeadsSigma-AldrichA2220IP
Antibodygamma H2A.X (phospho s139)abcamab2893IF(1:3000), WB (1:1000)
AntibodyGoat Anti-Rabbit IgG H and L (HRP)abcamab6721WB (1:10000)
AntibodyHistone H3abcamab1791WB (1:5000)
AntibodyHistone H3 (acetyl K56)abcamab76307WB (1:1000)
AntibodyHSC 70Snata-Cruz Biotechnologysc-7298WB (1:1000)
AntibodyKu80Cell Signaling#2180WB (1:1000)
AntibodyMRE11abcamab214WB (1:1000)
Antibodyphospho-ATM (Ser1981)Cell Signaling#5883WB (1:1000)
Antibodyphospho-Histone H2A.X (Ser139)Millipore05–636IF (1:1500)
AntibodyRabbit Anti-Mouse IgG H and L (HRP)abcamab97046WB (1:10000)
AntibodySIRT6abcamab62739WB (1:1000)
AntibodyTubulinMerckMAB1637WB (1:1000)
Peptide, recombinant proteinSIRT1 HumanPROSPECPRO-1909DNA binding assay
Peptide, recombinant proteinSIRT3 HumanPROSPECPRO-462DNA binding assay
Peptide, recombinant proteinSIRT5 HumanPROSPECPRO-1774DNA binding assay
Peptide, recombinant proteinSIRT6 HumanPROSPECPRO-282DNA binding assay

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