Abstract
The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.
Highlights
The hINO80 Complex Catalyzes ATP-dependent Nucleosome Sliding— Previous studies revealed that the S. cerevisiae INO80 complex possesses both DNA- and nucleosome-activated ATPase and ATPdependent nucleosome sliding activities
To determine whether the hINO80 complex could catalyze ATP-dependent nucleosome mobilization, anti-FLAG agarose eluates from FLAG-hIes2 expressing HeLa cells were mixed with mononucleosomes reconstituted on a 32P-labeled, 216-base pair DNA fragment in the presence of ATP or a mixture of ATP and ATP␥S, a potent inhibitor of many ATPases
The hINO80 complex contains five additional proteins that appear to lack yeast orthologs; these include the b-ZIP domain-containing Amida protein, the forkhead-associated domain-containing MCRS1 protein, the NFRKB protein, and proteins encoded by the FLJ90652 and FLJ20309 ORFs
Summary
The hINO80 Complex Catalyzes ATP-dependent Nucleosome Sliding— Previous studies revealed that the S. cerevisiae INO80 complex possesses both DNA- and nucleosome-activated ATPase and ATPdependent nucleosome sliding activities. To investigate the possibility that the mammalian INO80-related complex possesses similar activities, we assayed anti-FLAG agarose eluates from FLAG-hIes2-expressing HeLa cells for ATPase and nucleosome remodeling activities. The hINO80 complex, immunopurified from FLAG-hIes2-expressing cells, catalyzed ATP hydrolysis in a reaction that was strongly dependent on the addition of DNA or nucleosomes.
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