Fig. 4.

Intranucleosome cross-linking inhibits nucleosome mobilization by SWI/SNF. Nucleosomes cross-linked via the N-terminal tail domains of H2A and un-cross-linked controls were remodeled by incubation with 1 nM SWI/SNF, and translational positions were analyzed on 4% polyacrylamide translational gels as described in Materials and Methods. (A and B) Remodeling of un-cross-linked and cross-linked nucleosomes assembled on 244-bp and 215-bp 5S DNA fragments, respectively (A) or on the 343-bp 601 DNA fragment (B). Lanes 1, 3, 5, 7, and 9 show un-cross-linked 244-bp control nucleosomes, while lanes 2, 4, 6, 8, and 10 contain cross-linked nucleosomes on the 215-bp fragment. Samples were incubated either in TE (lanes 1 and 2), in remodeling buffer alone (lanes 3 and 4), or in remodeling buffer and ATP alone (lanes 5 and 6), SWI/SNF alone (lanes 7 and 8), or both SWI/SNF and ATP (lanes 9 and 10). Nucleosome positions represented by each of the bands are shown beside the gel. Lanes 11 and 12 in panel A show un-cross-linked nucleosomes on the 215-bp 5S template before and after remodeling, respectively. (C) Exonuclease III mapping of nucleosome positions before and after RSC remodeling. Remodeling reactions with nucleosomes containing H2A G2C and the 343-bp 601 DNA fragment were stopped by addition of 300 ng CT DNA and then subjected to Exo III digestion for 5 min, and the products were analyzed by sequencing gel electrophoresis and autoradiography. Lanes 1 and 2, unremodeled control nucleosomes incubated with or without 0.25 unit of Exo III, respectively; lanes 3 and 4, remodeled control nucleosomes incubated with 0.25 or 0.5 unit of Exo III; lanes 5 and 6, unremodeled cross-linked nucleosomes incubated with or without 0.25 unit of Exo III; lanes 7 and 8, remodeled, cross-linked nucleosomes incubated with 0.25 or 0.5 unit of Exo III, respectively.