FIGURE 2.

Cooperative binding of PARP1 and ALC1 to nucleosomes. A, biotinylated DNA or mononucleosomes reconstituted with HeLa cell histones were immobilized on streptavidin beads and incubated for 5 min with PARP1 and NAD⁺, with or without ALC1 and ATP. After addition of competitor DNA, reactions were incubated for the indicated times. PARP1 and ALC1 in bound and unbound fractions was detected by Western blotting. B, nucleosome binding reactions were performed with the indicated concentrations of NAD⁺ and with or without benzamide as diagrammed in the figure. PARP1 and ALC1 in bound and unbound fractions was detected by Western blotting, and ³²P-PARylated PARP1 was detected by phosphorimaging of the same membrane used in the Western blots.