FIG. 2.

INO80 optimally requires 70 bp of extranucleosomal DNA to move nucleosomes to the center of DNA. (A to H) Nucleosome movement was tracked by site-directed mapping, with nucleosomes having a photoreactive group attached to amino acid residue 53 of histone H2B. Nucleosomes with various lengths of extranucleosomal DNA were used as described for Fig. 1, except that in these reaction mixtures salmon sperm DNA was added in place of unlabeled PCR DNA. The reaction mixtures contained 33 nM nucleosomes (based on octamer concentration), 40 nM INO80, and 800 μM ATP. The samples were analyzed as described in Materials and Methods, and the phosphorimages before (black) and after (gray) INO80 remodeling are overlaid to illustrate changes in nucleosome position. The number 0 refers to the original nucleosome position, and the other numbers to how many base pairs the nucleosome has shifted after INO80 remodeling. The lengths of extranucleosomal DNA in these experiments were as follows: no extranucleosomal DNA (0N0) (A), 20 bp DNA (0N20) (B), 33 bp (0N33) (C), 43 bp (0N43) (D), 53 bp (0N53) (E), 70 bp (0N70) (F), 109 bp (0N109) (G), and 168 bp (ON168) (H). The extent of nucleosomes shifted from the original position is indicated below each plot as a percentage of the initial nucleosomes that were moved. (I) The same remodeling reactions were stopped with γ-S-ATP and competitor DNA before loading on to a 5% PAGE gel (60:1, high resolution). The − and + symbols above each lane indicate whether INO80 was added, and the nucleosome nomenclature is the same as for panels A to H. (J) The extent of remodeling, based on changes in electrophoretic mobility indicated in panel I, was quantified and plotted relative to the length of extranucleosomal DNA.