Fig. 2.

PARylation converts PARP-1 from a chromatin architectural protein to a histone chaperone. (A and B) Representative binding curves and stoichiometry measurements of AM–PARP-1 to LE-Tri (●, solid lines) and NLE-Tri (▲, dashed lines). (C) Binding curves for PARP-1 and AM–PARP-1 to histones. H2A-H2B–PARP-1 (●, solid line), H3-H4–PARP-1 (■, dashed line), H2A-H2B–AM–PARP-1 (♢, solid line), and H3-H4–AM–PARP-1 (▿, dashed line) are shown. (D) Endogenous PARylated PARP-1 coimmunoprecipitates soluble histones. U2OS cells were treated with H₂O₂ in combination with PJ34 or gallotannin as indicated. PAR antibodies (P) or control IgG (I) were used in immunoprecipitation (IP) assays from soluble lysates. Bound proteins were detected by immunoblotting with a mixture of PARP-1 and PAR antibodies (Top) and H2B and H4 antibodies (Middle and Bottom). (E) AM–PARP-1 does not disassemble nucleosomes. Labeled Nuc165 with PARP-1 and 30Nick DNA was incubated with increasing amounts of NAD⁺. Samples were analyzed by native PAGE and visualized by gel FRET between H2B (cyanine 5 633/670) and H4 (Alexa488 488/520). Lane 2 contains nucleosomes alone; lane 3 contains nucleosomes with PARP-1 and 30Nick DNA; and lanes 4–8 contain nucleosomes with PARP-1, 30Nick DNA, and 0.1, 1, 10, 20, and 40 μM NAD⁺, respectively. FRET between histones H2B and H4 is observed in all lanes, indicating that nucleosomes remain intact throughout.