Abstract

ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement.

Highlights

  • DNA is packaged into nucleosomes, which are positioned by ATP-dependent chromatin remodelers

  • Ies2 was needed for Arp5-Ies6 association, as deletion resulted in the loss of the Arp5-Ies6 module within the INO80 complex (Fig. 1A)

  • Deletion of Arp5-FLAG wild-type (ARP5) resulted in an undetectable amount of Ies6 protein in whole cell extracts, whereas no substantial change in Ies6 was observed in ies2⌬ cells (Fig. 1B)

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Summary

Background

DNA is packaged into nucleosomes, which are positioned by ATP-dependent chromatin remodelers. Results: The Arp subunit of the INO80 complex that lacks unique insertion domains stimulates ATP hydrolysis without facilitating nucleosome sliding. Ectopic addition of the wild-type Arp5-Ies module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. The addition of mutant Arp lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding These results define the requirements of Arp5-Ies assembly,. Previous research has demonstrated that the large multisubunit INO80 complex influences diverse DNA-templated processes through subunit specification of distinct chromatin remodeling activities [3, 11]. The insertion region of mammalian Ino is needed for ACTR5-INO80C module (Arp5-Ies in yeast) assembly within the INO80 complex [25], its requirement for the yeast complex has not previously been reported.

The abbreviations used are
Experimental Procedures
Results
B Ino80-F Arp5-F Ies6-F
Discussion
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