Activation of the SNF2 family ATPase ALC1 by poly(ADP-ribose) in a stable ALC1·PARP1·nucleosome intermediate

Abstract

The human ALC1/CHD1L oncogene encodes an SNF2 family ATPase with a macrodomain that binds poly(ADP-ribose) (PAR). We and others previously showed that ALC1 possesses a cryptic ATP-dependent nucleosome remodeling activity that is potently activated in the presence of PARP1 and NAD(+), its substrate for PAR synthesis. In this work, we dissected the mechanism by which PARP1 and NAD(+) activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Taken together, our findings are consistent with the model that PAR present on PARylated PARP1 acts as an allosteric effector of ALC1 nucleosome remodeling activity.

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.

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.

FIGURE 3.

The ALC1 macrodomain protects PAR chains from ∼ 3 to more than 20 ADP-ribose units from digestion by PARG. A, diagram showing the protocol used for PAR footprinting assays. B, reactions performed as diagrammed in A contained no PARG (lanes 1 and 5) or 1.5 ng (lanes 2 and 6), 5 ng (lanes 3 and 7), and 15 ng (lanes 4 and 8) PARG. C, reactions performed as diagrammed in A contained PARP1 and wild-type or mutant ALC1 without or with 5 ng of PARG. Marker lanes (M) show the total reaction products synthesized in a reaction containing nucleosomes and PARP1. Free ATP runs at the position indicated by the asterisk.

FIGURE 4.

Proposed pathway for ALC1 activation by PARP1 and NAD⁺. Unmodified PARP1 binds nucleosomes with high affinity. NAD⁺-dependent PARylation of PARP1 decreases its affinity for nucleosomes or DNA; thus, in the absence of ALC1 the nucleosome-PARylated PARP1 intermediate tends to dissociate. ALC1 and PARylated PARP1 bind cooperatively to nucleosomes, leading to formation of a stable, activated intermediate that can catalyze ATP-dependent nucleosome remodeling. PARP1*, PARylated PARP1; Nuc, nucleosome.