Figure 1.

Histone Serine ADPr Is Dependent on HPF1

(A) SILAC strategy to quantify core histone ADPr marks after DNA damage in WT and ΔPARP-1 U2OS cells (left) and a representative western blot of total protein poly-ADP-ribosylation prior to mixing light and heavy lysates from each SILAC experiment (right). Anti-GAPDH was used as a loading control.

(B) MS1 of a PARP-1-sensitive modified H2B peptide. The heavy peptide was derived from WT cells, and the light peptide was derived from ΔPARP-1 cells (both stimulated with H₂O₂). The inset (right) shows an ∼1:1 ratio (heavy/light) of a non-ADP-ribosylated peptide from the same experiment.

(C) Autoradiogram shows ADP-ribosylation of recombinant H3 by PARP-1 in the presence of HPF1.

(D) Autoradiogram shows ADP-ribosylation of two synthetic peptide variants corresponding to amino acids 1–21 of human H3.

(E) Autoradiogram shows histone tetramer ADP-ribosylation by PARP-1, PARP-2, or PARP-3 in the presence of HPF1.

(F) SILAC strategy to quantify core histone ADPr marks upon DNA damage in WT and ΔHPF1 U2OS cells (left) and a representative western blot of total protein poly-ADP-ribosylation levels prior to mixing light and heavy lysates from each SILAC experiment (right). Anti-GAPDH was used as a loading control.

(G) MS1 of an HPF1-sensitive H2B-modified peptide. The heavy peptide was derived from the WT cells, and the light peptide (very low intensity) was derived from ΔHPF1 cells (both were stimulated with H₂O₂). The inset (right) shows an ∼1:1 ratio (heavy/light) of a non-ADP-ribosylated peptide from the same experiment.

See also Figure S1.