Summary We know very little about the mechanism of INO80, how it disrupts nucleosomes and the factors governing its activity. We will take detailed “snapshots” of INO80 during nucleosome remodeling to find how INO80 and nucleosomes are moved during remodeling. A series of orthogonal approaches will be used to arrest INO80 remodeling at distinct stages and examine conformational changes in the core nucleosome and INO80. We will build on our recent observations of the motor domain being engaged at the H2A-H2B interface and persistently displacing DNA from this surface to find why displacement occurs, the factors that control displacement and whether this displacement weakens the interactions of H2A or H2A.Z dimers with the rest of the histone octamer or otherwise disrupts the nucleosome structure. Based on the proximity of Arp5 to nucleosomal DNA, we will test the premise of Arp5 as the “gatekeeper” regulating DNA traversing through the center of nucleosomes with wild type INO80 and mutant Arp5 in which either its histone or nucleosome binding regions have been deleted or mutated. We will also test whether the Arp8 module regulates Arp5 interactions with the acidic pocket of nucleosomes or nucleosomal DNA and if communication between these two domains is mediated by the Ino80 catalytic subunit. INO80 will be arrested at different stages in remodeling by limiting DNA translocations to specified distances, arresting with non-hydrolyzable ATP analogs, limiting linker DNA length and mutation of Arp8 and Arp5. We will probe the role of DNA sequence in INO80 remodeling because we observed coupling of ATPase activity to nucleosome movement being dramatically affected by the DNA sequence of the core nucleosome. We will find as suggested in these experiments if INO80 interactions and conformation varies depending on the DNA sequence bound by nucleosomes. In order to better examine the importance of DNA sequence in a “native” context, we will use yeast chromatin reconstituted with recombinant histones and simultaneously examine the differences of INO80 binding and remodeling with many thousands of nucleosomes, each with a different DNA sequence. We will use our expertise of mapping protein-DNA interactions in these genomic assays to sort with high precision the interactions of the INO80 subunits along with nucleosome movement, composition and structural features at ~bp resolution to provide a detailed analysis of each of these nucleosomes in a time resolved manner when remodeled. This approach will provide more insights into the DNA sequence specificity of INO80 and if there are “hot spots” for mobilizing/ destabilizing nucleosomes or exchanging H2A.Z in the yeast genome that doesn’t require additional factors. To confirm if INO80 behaves the same in vivo as in our in vitro assays, we will transfer several of these approaches to yeast cells so that we can measure chromatin dynamics in vivo with the same resolution as in vitro. We will compare how mutations i...