Dissecting the unique chaperone mechanism of the Rvb1/Rvb2 AAA+ ATPase complex

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PROJECT SUMMARY/ABSTRACT Multi-subunit protein machines are fundamental to nearly all biological processes, ranging from DNA replication to protein translation. Assembly of these protein machines requires processes that enable coordination of necessary interactions between subunits while preventing inappropriate ones. One way the cell manages to do this is by utilizing chaperones, a diverse group of proteins that assist in the non-covalent folding and unfolding, and assembly and disassembly of macromolecular structures. Rvb1 and Rvb2 are two essential AAA+ ATPases from S. cerevisiae that are proposed to act together (Rvb1/Rvb2) as an assembly chaperone for various multi- subunit complexes, including select chromatin remodelers. Rvb1 and Rvb2 are also associated with various cellular processes, including transcription, DNA-damage, and cell signaling, and their defects are strongly associated with multiple cancers, including hepatocellular carcinoma and acute myeloid leukemia. Yet how the Rvb1/Rvb2 complex functions as a chaperone is poorly understood. This proposal will explore Rvb1/Rvb2 function in the context of assembly and activity of the INO80 and SWR1 chromatin remodeling complexes. INO80 and SWR1 are chromatin remodelers composed of multiple subunits, including a Rvb1/Rvb2 hexamer, that use the energy from ATP hydrolysis to remodel nucleosome substrates. There is evidence for Rvb1/Rvb2 chaperone-like activity in the context of INO80 complex assembly. A proposed intermediate of INO80 assembly contains Rvb1/Rvb2 and exhibits enhanced Rvb1/Rvb2 ATPase activity. However, Rvb1/Rvb2’s role throughout complex assembly remains unclear. INO80 and SWR1 have similar overall architectures, but have unique subunits and nucleosome remodeling activities. Distinct interactions between Rvb1/Rvb2 and the insertion regions of a core subunit, Ino80p and Swr1p, may regulate unique complex assembly and activity of INO80 and SWR1, respectively. This proposal will (i) identify and characterize intermediates of the Rvb1/Rvb2-mediated INO80 assembly pathway and (ii) test whether and how Ino80p and Swr1p insertion regions regulate specific complex assembly and activity. This will be done using a combination of in vitro biophysical and biochemical techniques. The results will uncover the steps in INO80 and SWR1 assembly that are regulated by Rvb1/Rvb2 and in doing so identify major points of quality control in the assembly and function of these complexes in vivo. The principles derived from this study will be broadly applicable to understanding how the Rvb1/Rvb2 complex functions in other cellular processes and how defective Rvb1/Rvb2 functioning contributes to specific diseases. This research will be performed at UCSF under the direct supervision of Dr. Geeta Narlikar, an expert in using biochemical tools to dissect mechanism of chromatin regulators. The Narlikar lab and UCSF is equipped with all tools and resources necessary to accomplish the proposed research and training goals.