Fig. 1.

The N-terminus of Arp8, C-terminus of Arp4, and the HSA domain of Ino80 each associate with extranucleosomal DNA. a Top panel: Cartoon representation of the INO80 complex on nucleosomes of the sites where photoreactive nucleotides are incorporated by their distance in nucleotides from the dyad axis. Bottom panel shows photo-crosslinked INO80 subunits at each site on extranucleosomal DNA. b Crosslinking efficiencies of INO80 subunits at various nucleotide positions. Values are mean of ≥3 experiments, normalized to Ino80 at nt −58 without ADP. Error bars denote ± s.d. c Schematic outlining the protein-DNA crosslinking/peptide cleavage strategy used in d-f. Photo-crosslinked [³²P]-radiolabeled peptides resulting from cleavage at methionines (Met) or cysteines (Cys) are compared with custom-synthesized [³⁵S] peptide molecular weight markers. For photo-crosslinked Ino80 immobilized via the C-terminal FLAG and cleaved at arginines (ArgC protease), all FLAG-tagged fragments are identified by immunoblotting and are compared to photo-crosslinked fragments. d, e Schematics show the locations of Cys and Met in Arp8 (d) and Arp4 (e) plus the conserved actin-fold regions, non-actin insertions, and the regions that crosslink to DNA (highlighted by a black bar and red asterisk). Bottom panels are representative phosphorimages and the amount of CnBr or NTCB increases from left to right. Numbers on the left correspond to general protein molecular weight markers (in kDa), and on the right, correspond to custom-synthesized Arp8/Arp4 peptide markers, shown by their length in amino acids relative to the intact protein. f The region of Ino80 crosslinked to nt −111/−110 was mapped using ArgC protease. Cleavage sites are depicted as in d and e. Numbers on the left correspond to custom-synthesized markers (their molecular weights expressed in kDa), and on the right, indicate the lengths of the peptides resulting from ArgC cleavage, in number of amino acids from the C-terminus. Uncropped images showing the peptide-mapping fragments alongside custom-synthesized markers on the same gel are shown in Supplementary Fig. 2