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      Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM

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          Abstract

          Cryo-EM reveals the remodeling of RUVBL1-RUVBL2 ATPases by the client recruitment component in the HSP90 co-chaperone R2TP.

          Abstract

          The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2–containing complexes.

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          Structural basis for nucleosome remodeling by the INO80 complex

          DNA in the eukaryotic nucleus is packaged in the form of nucleosomes, ~147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP dependent chromatin remodelers1–3 such as the 15 subunit INO80 complex4. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, exchanging histone H2A.Z with H2A, and positioning +1 and -1 nucleosomes at promoter DNA5–8. A structure and mechanism for these remodeling reactions is lacking. Here we report the cryo-electron microscopy structure at 4.3Å resolution, with parts at 3.7Å, of an evolutionary conserved core INO80 complex from Chaetomium thermophilum bound to a nucleosome. INO80core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. A Rvb1/2 AAA+ ATPase hetero-hexamer is an assembly scaffold for the complex and acts as stator for the motor and nucleosome gripping subunits. The Swi2/Snf2 ATPase motor binds to SHL-6, unwraps ~15 base pairs, disrupts the H2A:DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5-Ies6 grip SHL-2/-3 acting as counter grip for the motor on the other side of the H2A/H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 core and entry DNA over a distance of ~90Å and packs against histone H2A/H2B near the acidic patch. Our structure together with biochemical data8 suggest a unified mechanism for nucleosome sliding and histone editing by INO80. The motor pumps entry DNA across H2A/H2B against Arp5 and the grappler, sliding nucleosomes as a ratchet. Transient exposure of H2A/H2B by the motor and differential recognition of H2A.Z and H2A may regulate histone exchange during translocation.
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            HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

            RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases. Copyright © 2010 Elsevier Inc. All rights reserved.
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              Structure and regulation of the human INO80-nucleosome complex.

              Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.
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                Author and article information

                Journal
                Sci Adv
                Sci Adv
                SciAdv
                advances
                Science Advances
                American Association for the Advancement of Science
                2375-2548
                May 2019
                01 May 2019
                : 5
                : 5
                : eaaw1616
                Affiliations
                [1 ]Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid, Spain.
                [2 ]Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK.
                Author notes
                [*]

                These authors contributed equally to this work.

                []Corresponding author. Email: ollorca@ 123456cnio.es
                Author information
                http://orcid.org/0000-0001-5705-0699
                Article
                aaw1616
                10.1126/sciadv.aaw1616
                6494491
                31049401
                c504aa19-dd81-441b-aeae-6bea493de682
                Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

                This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 23 November 2018
                : 16 March 2019
                Funding
                Funded by: doi http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 095605/Z/11/A
                Funded by: doi http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 095605/Z/11/Z
                Funded by: Spanish Ministry of Science, Innovation and Universities (MCIU/AEI), co-funded by the European Regional Development Fund (ERDF);
                Award ID: SAF2014-52301-R, SAF2017-82632-P
                Funded by: Spanish Ministry of Science, Innovation and Universities (MCIU/AEI), co-funded by the European Regional Development Fund (ERDF);
                Award ID: BFU2017-87316-P
                Funded by: Spanish Ministry of Science, Innovation and Universities (MCIU/AEI), co-funded by the European Regional Development Fund (ERDF;
                Award ID: BES-2015-071348
                Categories
                Research Article
                Research Articles
                SciAdv r-articles
                Molecular Biology
                Structural Biology
                Structural Biology
                Custom metadata
                Abigael Omaña

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