Figure 4.

DNase I footprinting of MENT reconstituted with trinuclesomes. (A) Reconstituted nucleosome trimers labelled with ³²P-ATP without MENT (lanes 1–8) or reconstituted with two molecules of MENT per nucleosome (lanes 9–16) were incubated with DNase I for 0 (1,9), 1 (2, 10), 2 (3, 11), 5 (4, 12), 10 (5, 13), 20 (6, 14), 30 (7, 15) and 40 (8, 16) min and analysed on a 6% polyacrylamide/urea gel. The molecular size markers (lane MW) represent ³²P-labelled DNA of pUC19 (GenBank accession no. L09137) and pEGFP-C3 (GenBank accession no. U57607) simultaneously digested with MspI restriction enzyme. (B–D) Magnified regions of the gel where MENT interferes with DNase I digestion. The molecular sizes of the DNA bands (bp) are indicated. On the left of panels B–D quantitation of DNase I footprints is shown. The extent of protection of each region in trinucleosome from DNase I without (heavy line) and with MENT (light line) was quantitated using ImageJ.