Recruitment of the INO80 Complex by H2A Phosphorylation Links ATP-Dependent Chromatin Remodeling with DNA Double-Strand Break Repair

Abstract

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

Chromatin disassembly and reassembly during DNA repair

“…1C). Alternatively, because arp8 mutants have a defect in DNA resection [78], INO80-dependent chromatin remodeling may help recruit the MRX complex to the DNA or may facilitate the ability of the MRX complex to mediate DNA resection on chromatin. In this scenario, chromatin disassembly may be the consequence of the resection machinery physically displacing histone proteins from the DNA -this is easy to imagine given the extensive contacts that are made between the DNA resection machinery and the DNA double-helix during the 5'-3' exonucleolytic attack.…”

“…In yeast lacking the INO80 remodeler, an approximate 50% reduction in the rate of DNA resection at the HO site has been observed [78]. These defects in DNA repair are fairly subtle and indicate that chromatin disassembly by either Asf1, INO80 or CAF-1 is clearly not essential to enable the DNA repair machinery to access and repair the DNA lesion.…”

INO80 subfamily of chromatin remodeling complexes

“…In a microarray assay, it was found that 150 out of 5602 yeast genes showed at least 2 fold change in mRNA level upon acute removal of INO80, with roughly equal numbers of up regulated and down regulated genes [17]. Several other global gene expression profiling experiments also showed that the INO80 complex regulates a specific set of gene both negatively and positively [9,47]. Two phosphate regulated genes, PHO5 and PHO84 were used to study the promoter regulation mechanism of INO80, and it was found that INO80 had a repressive effect on the PHO5 promoter and a stimulatory effect on the PHO84 promoter, suggesting that INO80 is required for proper regulation of these promoters [17].…”

“…Phosphorylated H2AX, referred to as γ-H2AX, is needed for the recruitment and/or retention of several DNA repair proteins [58]. Recent studies indicate that yeast γ-H2AX is also required for the recruitment of the chromatin remodeling complex INO80 to DSB sites [33,47,59], thus established a link between chromatin remodeling and DNA repair. Given that the ino80 mutant is hypersensitive to DNA damage agents [8,10], Morrison et al and van Attikum et al asked whether Ino80 is involved directly in the DNA damage processing.…”

“…Given that the ino80 mutant is hypersensitive to DNA damage agents [8,10], Morrison et al and van Attikum et al asked whether Ino80 is involved directly in the DNA damage processing. They found that transcriptional and checkpoint responses to DNA damage were normal in ino80 and arp8 mutants by comparing specific genes and global expression patterns, suggesting that INO80.com participates in the DNA damage response independent of transcription [33,47]. Moreover, an HO endonuclease-induced DSB system and ChIP assays were used to show that Ino80, Arp5, Arp8, and Rvbs are recruited directly to the HO-induced DSB in yeast [33,47,59].…”