Abstract
A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.
Highlights
During DNA replication, genome integrity is vulnerable, since various factors––such as chemical agents, proteins tightly bound to DNA or specific DNA structures––could act as obstacles and stall advancing replication forks
This work aimed to examine the requirements for the human INO80 chromatin remodeler in both unperturbed replication and in recovery from replication stress, motivated by the limited data of its participation in DNA replication in mammalian cells and the disparate conclusions reached by yeast studies
To elucidate the mechanism of action of INO80 in DNA replication, we systematically investigated the involvement of the complex during unchallenged replication and under conditions of replication stress by following the effects of its depletion on cell survival, the fate of individual replication forks, S-phase checkpoint activation and the consequences of prolonged fork stalling
Summary
During DNA replication, genome integrity is vulnerable, since various factors––such as chemical agents, proteins tightly bound to DNA or specific DNA structures––could act as obstacles and stall advancing replication forks. The basic unit of chromatin is the nucleosome, which is composed of 147 bp of DNA wrapped around a histone octamer comprising a tetramer of (H3– H4) flanked by two dimers of H2A–H2B. During replication the chromatin structure undergoes major reorganization as nucleosomes are disassembled ahead of the replication fork and reassembled behind it. An increasing body of evidence suggests that replicative helicases, histone chaperones and chromatin remodelers form an assembly line at the replication forks [4]. This necessitates the study of the contribution of ATP-dependent chromatin remodeling complexes in the processes of chromatin replication and maintenance of genome stability [4,5,6]
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