FIGURE 1.

Rapid formation of a benzamide-resistant intermediate in ALC1 activation. A, diagram describing the protocol and positioned nucleosome substrate with an HhaI site on 216-bp 601-lat Gal4 DNA fragment used for benzamide challenge nucleosome remodeling assay. The asterisk indicates ³²P-labeled DNA end. B, Naked DNA (lane 1) or positioned nucleosomes (lanes 2–14) were incubated with HhaI in the absence (lane 1) or presence (lanes 2–14) of PARP1. Where indicated, PARP1 inhibitor benzamide was added to reactions at the times shown in the figure. One minute later, ALC1 was added, and remodeling reactions proceeded for an additional 60 min, and DNA or nucleosomes were monitored for HhaI restriction enzyme accessibility. C, quantitation of experiment shown in B. D, kinetics of PAR synthesis. Reactions with [³²P]NAD⁺ were performed according to the protocol shown in A, except that ALC1 was omitted. PAR synthesized during the reactions was purified, analyzed on denaturing polyacrylamide gels, and visualized on a phosphorimager (left panels). Marker lane (M) contains [γ-³²P]ATP. The graph on the right shows traces of lanes 4–7 generated using ImageQuant TL software.