The integrated stress response induces R-loops and hinders replication fork progression

The integrated stress response (ISR) allows cells to rapidly shut down most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within one hour, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or re-activate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops) which interfere with DNA replication. Histone depletion alone induces R-loops and compromises DNA replication. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses. SIGNIFICANCEThe integrated stress response has long been explored regarding its immediate impact on protein synthesis. Translational shutdown represents an indispensable mechanism to prevent the toxicity of misfolded proteins and virus infections. Our results indicate that the shutdown mechanisms reach far beyond translation and immediately interfere with DNA synthesis as well. ISR depletes cells of new histones which induce accumulation of DNA:RNA hybrids. The impairment of DNA replication in this context supports cell survival during stress. Our work provides a link between the ISR and another subject of active research, i. e. the regulatory network of DNA replication forks. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/987131v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1e65182org.highwire.dtl.DTLVardef@1f157cforg.highwire.dtl.DTLVardef@1bc0c59org.highwire.dtl.DTLVardef@ceb234_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG