Figure 6.
mH2A1.1 down-regulates PARP-1 enzymatic activity. (A) Silver staining of the purified wild-type macroH2A1.1 (e-mH2A1.1) and the mutant (e-mH2A1.1-mut) nucleosomes used for measurement of PARP-1 enzymatic activity. The complexes were isolated using 100 mM NaCl. The bands corresponding to PARP-1, e-mH2A1.1, and the conventional core histones are indicated. (M) Protein molecular mass marker. (B) Kinetic analysis of PARP-1 activity of purified mH2A1.1 complex (left panel), purified mH2A1.1 mutant complex (middle panel), and recombinant PARP-1 (right panel) in the presence of 200 nM 32P-NAD+. To measure the auto-ADP-ribosylation activity of the recombinant PARP-1, an amount of nucleosomes (100 ng) and recombinant PARP-1 equal to that in the mH2A1.1 complexes was used. Note the close auto-ADP-ribosylation kinetics of the recombinant PARP-1 and the PARP-1 in the mH2A1.1-mut.com. (C) Quantification of the data shown in B. Note the dramatic difference in the auto-ADP-ribosylation kinetics of PARP-1 associated with the wild-type and the mutated mH2A1.1 complexes. (D) Twelve percent SDS-PAGE of the purified e-mH2A1.1 octamers and associated PARP-1. e-mH2A1.1 nucleosome complexes were loaded on a hydroxyl apatite column, and after washing with 0.65 M NaCl the remaining proteins were eluted with 2 M buffered solution of NaCl. To purify PARP-1 from the histone octamers, the 2 M NaCl eluate was supplemented with 1 M urea and passed through an agarose-nickel column. (Lanes 1,2) Molecular mass marker and conventional histone octamer as a control. (Lane 3) Tthe protein composition of the 2 M NaCl eluate. (Lane 4) The purified PARP-1. (E) Kinetic analysis of PARP-1 activity associated with in vitro reconstituted nucleosomes containing either conventional core histones (lanes 2–5) or purified e-mH2A1.1 core histones (lanes 6–9). The samples were incubated with 32P-NAD+ and run on 12% PAGE containing SDS. Lane 1 contains 32P-NAD+ only.