Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

Abstract

When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates

“…INO80 has a role in removing nucleosomes from around double-strand DNA breaks (Li and Tyler, 2016, Tsukuda et al., 2005) and is also involved in resolving replication-transcription conflicts (Poli et al., 2016). Loss of both INO80 and chromatin accessibility complex (CHRAC) remodelers in vivo causes reduced nucleosome accessibility to MNase around replication forks in methyl methanesulfonate (MMS) (Lee et al., 2015), consistent with the idea that these remodelers play some role in disrupting nucleosomes during replication through damaged DNA. Our results suggest that INO80 may also play a role in normal replication.…”

“…In contrast to FACT, Nhp6 is required at much higher levels than replication factors to exert its effect on replication, suggesting that it may act distributively on chromatin. That INO80 and CHRAC contribute to changes in nucleosome accessibility specifically around replication forks suggests that they may be targeted to replication forks (Lee et al., 2015). Recent work has shown that INO80 is recruited to replication forks in human cells through interaction with ubiquitylated H2A aided by BRCA1-associated protein-1 (BAP1) (Lee et al., 2014), but it is unclear whether or how INO80 is being recruited to forks in our system.…”

Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes

“…Despite extensive biochemical characterization of ATP-dependent nucleosome loading and remodeling (Bartholomew, 2014; Clapier and Cairns, 2009) and the apparent role of remodeling enzymes during DNA replication (Biswas et al, 2008; Collins et al, 2002; Lee et al, 2015; Poot et al, 2004; Vincent et al, 2008), it has remained unclear to what extent nucleosome assembly is an ATP-dependent process in vivo . Several studies have shown that Chd1 and ISWI proteins require histone chaperones during the process of nucleosome assembly (Fyodorov and Kadonaga, 2002; Ito et al, 1997; Lusser et al, 2005; Torigoe et al, 2013; Tsukiyama et al, 1999).…”

Recovery from the DNA Replication Checkpoint

“…Indeed, evidence indicates that local action of the Slx4/Rtt107 complex at replication forks is complementary to the global activity of the Pph3/Psy2 Rad53 phosphatase [40]. In addition, chromatin remodeling factors Ino80 and Isw2—demonstrated to promote chromatin accessibility—attenuate S-phase checkpoint signaling and promote the recovery of stalled replication forks, although the mechanism by which this is accomplished in not known [41,42]. …”