(A) 53BP1 binds H4K20me1, as determined by a SILAC-based histone peptide pull-down approach. Proteins are plotted according to their SILAC-ratio in the “forward” (x-axis; unmodified peptide incubated with “light” extract, modified peptide incubated with “heavy” extract) and “reverse” (y-axis; unmodified peptide incubated with “heavy” extract, modified peptide incubated with “light” extract) experiment. Specific interactors should lie close to the diagonal in the upper right quadrant. Background binders cluster together in the bottom left part of the graph, showing a 1:1 ratio in both experiments.
(B) 53BP1 binds H4K20me2, as determined by a SILAC-based histone peptide pull-down approach. Plot is as described in (A).
(C) PR-Set7 recruitment to a DNA damage site precedes that of 53BP1. YFP-PR-Set7, green; mCherry-53BP1, red; representative data of n=8 cells is shown; scale bar, 5 μm.
(D) Cells treated with siRNA against PR-Set7 exhibit similar 53BP1 recruitment to a DNA damage site as in (B), upon transient expression of siRNA resistant WT PR-Set7. YFP-PR-Set7 siR, green; mCherry-53BP1, red; representative data of n=7 cells is shown; scale bar, 5 μm
(E) In contrast to (C), siRNA-PR-Set7 treated cells are devoid of detectable 53BP1 recruitment to a DNA damage site upon transient expression of the catalytically inactive PR-Set7 mutant (R265G). YFP-PR-Set7 R265G, green; mCherry-53BP1, red; representative data of n=8 cells is shown; scale bar, 5 μm.
(F) PR-Set7 is required for 53BP1 binding to the DNA damage site. Cells treated with PR-Set7 siRNA for 3 days were transfected with mCherry-53BP1. YFP-PR-Set7, green; mCherry-53BP1, red; representative data of n=10 cells is shown; scale bar, 5 μm.
(G) Recruitment of endogenous 53BP1 to a DNA damage site requires PR-Set7. U2OS cells expressing YFP-PCNA were mock treated or transfected for 3 days with siRNA against PR-Set7. Cells were fixed 6 min after the laser pulse and subjected to immunofluorescence labeling of endogenous 53BP1. YFP-PCNA, green; anti-53BP1, red; Hoechst DNA labeling, blue; arrows indicate the irradiated site; representative data of n=6 (U2OS) or n=10 (U2OS siRNA PR-Set7) cells is shown; scale bar, 5 μm.