1. Biochemistry and Chemical Biology
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Serine is the major residue for ADP-ribosylation upon DNA damage

  1. Luca Palazzo
  2. Orsolya Leidecker
  3. Evgeniia Prokhorova
  4. Helen Dauben
  5. Ivan Matic  Is a corresponding author
  6. Ivan Ahel  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. Max Planck Institute for Biology of Ageing, Germany
Research Advance
Cite as: eLife 2018;7:e34334 doi: 10.7554/eLife.34334
4 figures, 1 table and 1 additional file

Figures

HPF1-dependent Ser-ADPr is the major form of ADPr upon genotoxic stress.

(A) Control, ARH3 KO (ARH3−/−), HPF1 KO (HPF1−/−), and PARP1 KO (PARP1−/−) U2OS cells were treated with 2 mM H2O2. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr, mono-ADPr, PAR, PARP1, ARH3, H3, and HPF1 antibodies. Additionally, Ponceau-S staining was used as loading control. (B) Control, ARH3 KO (ARH3−/−) and two independent clones of HPF1 KO (HPF1−/−−1 and HPF1−/−−2) U2OS cells were treated with 2 mM H2O2. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr, PAR, ARH3, and HPF1 antibodies. Ponceau-S staining was used as loading control. (C) Control, ARH3 KO (ARH3−/−), and HPF1 KO (HPF1−/−) U2OS cells were treated with 2 mM MMS. After the induction of DNA damage, the cells were left to recover from genotoxic stress for the indicated time points. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr, ARH3, and HPF1 antibodies. Ponceau-S staining was used as loading control. (D) Control and HPF1 KO (HPF1−/−) HEK293 cells were treated with 2 mM H2O2. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr, ARH3, and HPF1 antibodies. Ponceau-S staining was used as loading control.

https://doi.org/10.7554/eLife.34334.002
HPF1-dependent ADPr is resistant to hydroxylamine.

(A) Autoradiogram shows serine ADPr of two synthetic peptides (wild type (WT) or Ser10Ala (S10A) mutant) corresponding to amino acids 1–21 of human H3 by wild type PARP1 or PARP1 E988Q in the presence of HPF1, with or without treatment with 1M NH2OH (hydroxylamine). Imperial Blu staining was used to show equal loading of samples. (B) Autoradiogram shows auto-ADPr of PARP1 E988Q (at glutamate residues) and the effect of the treatment with 1M NH2OH. Imperial Blue staining was used to show equal loading of samples. (C) Whole cell extracts were prepared from pre-damaged U2OS wild type or HPF1 KO (HPF1−/−) cells. Extracts were either left untreated or treated with 1M hydroxylamine (NH2OH) for 3 hr prior to separation on SDS-PAGE gel and immunoblotting with pan-ADPr, PAR or HPF1 antibodies. Ponceau-S staining was used as loading control. (D) Whole cell extracts were prepared from U2OS wild type or HPF1 KO (HPF1−/−) cells following treatment with 2 mM H2O2 for 10’. Extracts were either left untreated or treated with 1M hydroxylamine (NH2OH) for 3 hr prior to separation on SDS-PAGE gel and immunoblotting with pan-ADPr, PAR, H3 or HPF1 antibodies. Ponceau-S staining was used as loading control.

https://doi.org/10.7554/eLife.34334.003
Serine six is the main ADPr site of histone H2B induced by DNA damage.

(A) Control and HPF1 KO (HPF1−/−) HEK293 cells were transfected or not with Flag-H2B wild type (wt) and Flag-H2B Ser6Ala mutant construct (S6A). 24 hr post-transfection, cells were treated with 2 mM H2O2. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr, Flag, and HPF1 antibodies. Ponceau-S staining was used as loading control. The black star marks the ADP-ribosylated Flag-tagged H2B protein in the whole cell wild type extract, which is absent in other extracts. (B) Flag-tagged H2B wild type (wt) and Ser6Ala mutant (S6A) were immunoprecipitated (IP) by using anti-Flag antibody from the lysates generated in Figure 3A. IPs were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr and Flag antibodies. Ponceau-S staining was used to stain light chains of Immunoglobulins (IgG) as loading control of the IP.

https://doi.org/10.7554/eLife.34334.004
Serine 10 and serine 28 are the main ADPr sites of histone H3 induced by DNA damage.

(A) HEK293 cells were transfected or not with Flag-H3.1 (Flag-H3) wild type (wt), Flag-H3.1 Ser10Ala (S10A), Flag-H3.1 Ser28Ala (S28A), and Flag-H3.1 Ser10Ala Ser28Ala double mutant (S10A S28A) constructs. 24 hr post-transfection, cells were treated with 2 mM H2O2. After treatment/recovery, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr and Flag antibodies. Ponceau-S staining was used as loading control. (B) Flag-tagged H3.1 (Flag-H3) wild type (wt), Ser10Ala (S10A), Ser28Ala (S28A), and Ser10Ala Ser28Ala double mutants (S10A S28A) were immunoprecipitated (IP) by using anti-Flag antibody from the lysates generated in Figure 4A. IPs were separated by SDS-PAGE, analysed by western blot and probed for pan-ADPr and Flag antibodies. Ponceau-S staining was used to stain light chains of immunoglobulins (IgG) as loading control of the IP.

https://doi.org/10.7554/eLife.34334.005

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
cell line (Homo sapiens)U2OSATCCHTB-96,
RRID:CVCL_0042
cell line (Homo sapiens)HEK293ATCCCRL-3216,
RRID:CVCL_0063
cell line (Homo sapiens)U2OS ARH3 KOFontana et al., 2017
cell line (Homo sapiens)U2OS HPF1 KOGibbs-Seymour et al. (2016)
cell line (Homo sapiens)U2OS PARP1 KOGibbs-Seymour et al. (2016)
cell line (Homo sapiens)HEK293 HPF1 KOGibbs-Seymour et al. (2016)
antibodyanti-PAR (rabbit polyclonal)Trevigen
(Gaithersburg, MD, US)
4336-BPC-100,
RRID:AB_2721257
WB 1:1000
antibodyanti-pan-ADP-ribose
(rabbit monoclonal)
Millipore (Billerica, MA, US)MABE1016,
RRID:AB_2665466
WB 1:1500
antibodyanti-mono-ADP-ribose
(rabbit monoclonal)
Millipore (Billerica, MA, US)MABE1076,
RRID:AB_2665469
WB 1:1000
antibodyanti-PARP1 [E102]
(rabbit monoclonal)
Abcam (Cambridge, UK)ab32138,
RRID:AB_777101
WB 1:1000
antibodyanti-histone H3, CT, pan
(rabbit polyclonal)
Millipore (Billerica, MA, US)07–690,
RRID:AB_417398
WB 1:2000
antibodyanti-ARH3/ADPRH (rabbit polyclonal)Atlas Antibodies
(Stockholm, Sweden)
HPA027104,
RRID:AB_10601330
WB 1:1000
antibodyanti-HPF1 (rabbit polyclonal)Gibbs-Seymour et al. (2016)WB 1:1000
antibodyanti-Flag HRP-conjugated
(mouse monoclonal)
Sigma-Aldrich
(St. Louis, MO, US)
A8592,
RRID:AB_439702
WB 1:5000
antibodyanti-Flag M2 agarose-conjugated
(mouse monoclonal)
Sigma-Aldrich
(St. Louis, MO, US)
A2220,
RRID:AB_10063035
IP
recombinant DNA reagentpDONR221 (Gateway vector)Thermo Fisher Scientific
(Waltham, MA, US)
12536017
recombinant DNA reagentpDEST C3X (Gateway vector)otherLaboratory of Fumiko Esashi
recombinant DNA reagentFlag-H2B wt (plasmid)This paperProgentiors: pDONR221-H2B;
Gateway vector:pDEST C3X
recombinant DNA reagentFlag-H3.1 wt (plasmid)This paperProgentiors: pDONR221-H3.1;
Gateway vector:pDEST C3X
recombinant DNA reagentFlag-H2B S6A (plasmid)This paperMade from Flag-H2B wt by
site-directed mutagenesis
recombinant DNA reagentFlag-H3.1 S10A (plasmid)This paperMade from Flag-H3.1 wt by
site-directed mutagenesis
recombinant DNA reagentFlag-H3.1 S28A (plasmid)This paperMade from Flag-H3.1 wt by
site-directed mutagenesis
recombinant DNA reagentFlag-H3.1 S10A S28A (plasmid)This paperMade from Flag-H3.1 S10A by
site-directed mutagenesis
peptide, recombinant proteinHuman PARP1Trevigen
(Gaithersburg, MD, US)
4668–02 K-01
peptide, recombinant proteinHuman PARP1 E988QFontana et al., 2017
peptide, recombinant proteinHuman HPF1Gibbs-Seymour et al. (2016)
peptide, recombinant proteinHuman histone H3
fragment (1-21) wt
Bonfiglio et al., 2017a
peptide, recombinant proteinHuman histone H3
fragment (1-21) S10A
Bonfiglio et al., 2017a
chemical compound, drugOlaparibCayman Chemical
(Ann Arbor, MI)
10621
chemical compound, drugADP-HPD, dihydrate,
ammonium salt
Calbiochem
(La Jolla, CA)
118415
chemical compound, drugHydrogen peroxideSigma-Aldrich
(St. Louis, MO, US)
H1009
chemical compound, drugMethyl methanesulfonateSigma-Aldrich
(St. Louis, MO, US)
129925
chemical compound, drugHydroxilamineSigma-Aldrich
(St. Louis, MO, US)
438227

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