Structural biochemistry of the 180 kDa Arp8-module of INO80 defines its function during chromatin remodelling

Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180 kDa Arp8-module of S. cerevisiae INO80 and establish its role as an extranucleosomal DNA binding element. The actin-fold of Arp8 engages N-actin in a novel “side-to-front” interaction and specifies thereby recruitment of the Arp4-N-actin heterodimer to the segmented, “two plug” scaffold of the helical HSA domain of Ino80. The HSA domain spans a distance over 120 Å and provides a binding platform for extranucleosomal DNA, which is required for nucleosome sliding and genome-wide nucleosome positioning. Together with the recent cryoEM structure of INO80Core-nucleosome complex, our results provide insights into the mechanism by which INO80 senses 40 bp extranucleosomal entry DNA to conduct highly processive chromatin remodelling. Overall design: There are 10 samples analyzed in total. Within those 10 samples we used Drosophila histones. Samples consist of the INO80 WT protein and three mutants. Those 10 samples also contain replicates. The salt gradient dialysis samples are used as control samples.