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.
Highlights
The compaction of DNA into chromatin in eukaryotes provides advantages for stability of the genetic material and problems for processes such as repair of DNA damage.systems have evolved to deal with DNA damage within the context of nucleosomes
We show that the human Ino80 core complex is inhibited by IP6 and determine that the basis of this inhibition is a reduction in nucleosome affinity
Previous work on yeast and human Ino80 complexes has used endogenous protein extracted from cells using a tagged Ino80 subunit [17,18]
Summary
Systems have evolved to deal with DNA damage within the context of nucleosomes. Nucleosomes are remodelled by sliding them away from damage sites to allow access by the repair machinery. Such chromatin remodellers can be simple single subunit proteins such as Chd or more complex, multi-subunit systems such as RSC or Ino80 [1]. Single subunit systems are able to slide nucleosomes so it is something of a mystery why such complicated, multi-subunit machines exist that appear to perform similar functions. The Ino family of remodellers contain a conserved core of subunits together with around half a dozen additional proteins that are species-specific [1].
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