UCSF

The fundamental basis of gene regulation is the physical accessibility of genetic material to downstream machinery that convert DNA into proteins. In eukaryotes, accessibility to the genome is ultimately regulated by the structure of the nucleosome—the fundamental unit of chromatin that is comprised of 147 base pairs of DNA tightly wrapped around a core of eight histone proteins. Of the many factors that affect nucleosome structure, ATP-dependent chromatin remodeling enzymes are unique in their ability to convert the chemical energy of ATP into physical changes in the nucleosome that would otherwise be energetically disfavorable. Of these remodelers, the INO80 remodeling enzyme is a 15-subunit complex from yeast that is unique both in its modular structure and its critical roles at sites of genomic instability such as double strand breaks and replication forks. However, very little is known about how the INO80 machinery is assembled or how it achieves specificity in nucleosome movement. In the first half of this thesis, we explore the potential roles of Rvb1 and Rvb2 (Rvbs), two poorly understood AAA+ ATPases that are integral components of the INO80 complex, as well as other multi-subunit complexes in the cell. While the Rvbs were traditionally thought of as DNA helicases, we have new evidence for their role as protein chaperones. We discover the first protein client of Rvbs, a small insertion region of the Ino80 ATPase (Ino80INS), which robustly stimulates the ATPase activity of the Rvbs while promoting dodecamerization of the normally hexameric Rvbs. Using a combination of structural methods including crosslinking mass spectrometry, native mass spectrometry, cryo-EM and integrative modeling, we find that two Ino80INS molecules bind asymmetrically and adjacent to one another along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients like Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly. In the second half of the thesis, we explore the molecular mechanism of nucleosome movement by INO80. While it is known that INO80 can center nucleosomes on a short piece of DNA, it is unknown how the motor accomplishes this task. Using a combination of enzymology and single molecule FRET, we find that INO80 exhibits a switch-like response to flanking DNA length, largely regulated by subcomplex of INO80 called the Nhp10 module. We also find that remodeling by INO80 is extremely processive: once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without re-assessing flanking DNA length, and can change the direction of nucleosome sliding without dissociation. Taken together, these findings provide unprecedented insight into INO80’s lifetime in the nucleus: from its assembly by Rvb1/Rvb2 to its action on chromatin.