title: Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex.
abstract: We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.
authors.affiliation: Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Willhoft
authors.affiliation: Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: Rohan
authors.initials: R
authors.lastname: Bythell-Douglas
authors.affiliation: Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: McCormack
authors.affiliation: Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK D.Wigley@imperial.ac.uk.
authors.forename: Dale B
authors.initials: DB
authors.lastname: Wigley
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009707:Nucleosomes / Q000187:drug effects / Q000378:metabolism; D010833:Phytic Acid / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009707:Nucleosomes; D021122:Protein Subunits; D011994:Recombinant Proteins; D010833:Phytic Acid; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C524421:INO80 protein, human