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Fenn, Sebastian (2011): Structural biochemistry of the INO80 chromatin remodeler reveals an unexpected function of its two subunits Arp4 and Arp8. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The INO80 complex is a chromatin remodeler involved in diverse nuclear processes like transcriptional regulation, replication fork progression, checkpoint regulation and DNA double strand break repair. In the yeast S. cerevisiae the complex consists of 15 subunits with a total molecular mass of about 1.2 MDa. Knowledge about the atomic structure and molecular architecture of the entire complex is scarce. Similarly, an understanding for the roles of the individual subunits of the complex is mostly lacking. Especially the function of actin and the actin related proteins Arp4 and Arp8 which are found as monomeric components of INO80 and other chromatin remodelers is poorly understood. The goal of this study was to elucidate the functional architecture of the INO80 complex by using a hybrid methods approach. Different structural techniques such as X-ray crystallography, small angle X-ray scattering (SAXS) and electron microscopy (EM) were combined to achieve this goal. Additionally, various functional assays to study the biochemical properties of the actin related proteins and their interaction with actin were employed. In a set of primary experiments expression and purification protocols for seven individual INO80 components, namely Arp4, Arp5, Arp8, Ies4, Ies5, Ies6 and Nhp10 could be established. Additionally, four subcomplexes containing more than one protein, namely Rvb1-Rvb2, Nhp10-Ies5, Nhp10-Ies5-Ies3 and Arp5-Ies6 were purified. Thereby two previously unknown interactions between the INO80 subunits Nhp10 and Ies5, as well as Arp5 and Ies6 could be identified. Subsequently, the newly identified complexes of Nhp10-Ies5-Ies3 and Arp5-Ies6 were studied with SAXS to obtain low resolution solution structures of both. On top of that the entire INO80 complex was purified endogenously from S. cerevisiae and studied by EM. Unfortunately, a three dimensional reconstruction of the remodeler could not be created. Crystallization attempts on all purified INO80 components were successful for the complex of Rvb1-Rvb2 and the actin related protein Arp4. Whereas the structure of Rvb1-Rvb2 could not be solved due to limited diffraction an atomic structure of ATP bound Arp4 at 3.4 Å resolution was obtained. Remarkably, Arp4 does not form filaments despite its high similarity to conventional actin. The lack of polymerization is confirmed by the SAXS structure of isolated Arp4 which indicates it to be monomeric and can be nicely explained on the basis of the crystal structure. Several loop insertions and deletions at positions which are crucial for contact formation within the actin filament, especially at the pointed end of the molecule, prevent Arp4 to engage in filament like interactions. Furthermore, the crystal structure of Arp4 reveals an ATP molecule to be constitutively bound to the protein. The lack of ATPase activity of Arp4 in contrast to actin can be explained with the help of the crystal structure as well. Several residues in the nucleotide clamping loops of Arp4 are divergent from actin leading to a tighter closure and better shielding of the phosphate moieties of the bound ATP from the environment. Most interestingly, Arp4 dramatically influences actin polymerization kinetics. Different fluorescence assays and in vitro TIRF microscopy were used to show that Arp4 is able to inhibit actin polymerization and to depolymerize actin filaments most likely by complex formation with monomeric ADP-actin via the barbed end. Its ability to inhibit actin filament nucleation without sequestering actin while still allowing ADP to ATP exchange within actin resembles the actin binding protein profilin. Arp8 was confirmed by SAXS measurements to be monomeric as well. It is able to sequester actin monomers and to slowly depolymerize actin filaments. Consistent with the formation of a discrete Arp4-Arp8-actin complex within the INO80 remodeler the effects of Arp4 on actin polymerization are further stimulated by Arp8. As both proteins reciprocally enhance their individual effects on actin it is likely that they help to maintain actin in a defined monomeric state within the INO80 chromatin remodeler. The data further suggest a possible assembly between actin and Arp4 via their barbed ends and a model how the Arp4-Arp8-actin complex is integrated into the INO80 chromatin remodeler. Taken together, the findings represent a remarkable advancement in the understanding of nuclear actin related proteins and nuclear actin biochemistry in general. Most excitingly, they indicate a link between chromatin remodeling and nuclear actin dynamics possibly giving chromatin remodeling complexes a role in the actin mediated large scale movement of chromatin.