Abstract
The integrated stress response (ISR) allows cells to rapidly shutdown 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 1 h, 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. R-loops accumulate upon histone depletion. 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.
Highlights
The integrated stress response (ISR) is widely known as a mechanism to shutdown the synthesis of most proteins when the cell suffers various stresses[1] through the activation of the following kinases
DNA replication is compromised shortly after ISR induction The ISR triggers a shutdown of protein synthesis, representing an emergency response to nutrient deprivation or proteotoxic stress
Despite the significant reduction in DNA replication progression following ISR stimulation, we did not observe a substantial increase in phosphorylation of Chk[1] or histone variant H2AX after 1 h (Supplementary Fig. S1N) or 4 h (Fig. 1q) as compared to gemcitabine, a well-established inducer of replicative stress[7] indicating that the ISR slows down replication forks without triggering a strong DNA damage response. These results suggest that the ISR triggers a shutdown in protein synthesis and imposes severe and immediate restrictions on DNA replication
Summary
The integrated stress response (ISR) is widely known as a mechanism to shutdown the synthesis of most proteins when the cell suffers various stresses[1] through the activation of the following kinases. Heme-regulated inhibitor (HRI) is triggered in the case of heme depletion in erythrocytes Each of these kinases triggers the phosphorylation of the alpha subunit of translation initiation factor eIF2 at Serine 512. This modification of eIF2 shuts down the translation of most mRNAs, with the exception of a few mRNAs that employ alternative mechanisms of translation initiation. One of these exceptions is the transcription factor ATF4, Besides gene expression, the replication of DNA represents an extreme demand on the cell with regard to metabolic activity and energy consumption.
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