The proposal aims at the study of the molecular mechanisms involved in the starting steps of the repair of the most severe type of DNA lesions, the double-stranded DNA breaks (DSBs). DSBs are caused by either environmental stress or by endogenous DNA metabolic processes. Inaccurate repair of DSBs leads to genomic instability, and thus, to cell death or cancerous malformations. Enzymes acting in DNA repair processes are required to access to DNA. This is, however, hampered by the compact and stable chromatin (DNA bound to histone proteins) structure. The project outlined in this proposal focuses on the investigation of the chromatin remodeling activity of human INO80 complex proposed to play role in the homologous recombination (HR) based repair of DSBs. The INO80 subfamily is the most recent addition to the SWI/SNF family of chromatin remodelers found in yeast, flies, plants and mammals. However, INO80 seems to be the most evolutionary conserved subfamily of chromatin remodeling enzymes, which has been found to play important roles in gene expression regulation, DNA replication and repair. INO80 exerts remodeling activities (e.g. nucleosome eviction, histone exchange) in a large enzyme complex. The human INO80 complex consists of a total of almost 20 essential and regulatory subunits. Precise mechanisms by which the human INO80 complex performs chromatin remodeling activities are still unrevealed, yet. We are addressing the key questions regarding the fate of the nucleosomes upon remodeling by the INO80 complex: Are they moved? If yes, how far? Are they replaced? If yes, when? By using a fluorescently labeled subunit of the INO80 complex and/or histone components of the nucleosome, an array of single molecule and ensemble approaches will provide insights into the functions of INO80 complex in chromatin dynamics, and its role in DNA damage repair processes.