Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation

ABSTRACT

Incidents that slow or stall replication fork progression, collectively known as replication stress, represent a major source of spontaneous genomic instability. Here, we determine the requirement for global protein biosynthesis on DNA replication and associated downstream signaling. We study this response side by side with dNTP deprivation; one of the most commonly used means to investigate replication arrest and replicative stress. Our in vitro interrogations reveal that inhibition of translation by cycloheximide (CHX) rapidly impairs replication fork progression without decoupling helicase and polymerase activities or inducing DNA damage. In line with this, protein deprivation stress does not activate checkpoint signaling. In contrast to the direct link between insufficient dNTP pools and genome instability, our findings suggest that replication forks remain stable during short-term protein deficiency. We find that replication forks initially endure fluctuations in protein supply in order to efficiently resume DNA synthesis upon reversal of the induced protein deprivation stress. These results reveal distinct cellular responses to replication arrest induced by deprivation of either nucleotides or proteins.

KEYWORDS:

• Cycloheximide
• translation
• stalled replication fork
• replication stress
• DNA damage

Acknowledgments

We thank members of the Helleday lab for discussions. The U2OS cells stably expressing H3.1 or H3.3 were a kind gift from Sophie Polo, Paris Diderot University, France.

Author summary

DNA replication is an essential nuclear process that ensures faithful duplication of the genome for its subsequent transfer to daughter cells during each round of the cell cycle. Accurate replication of genetic information must also be accompanied by restoration of the chromatin landscape on newly replicated DNA. Perturbations of these processes pose a potential threat to genome integrity as it is accompanied by DNA damage and subsequent mutations. A common way to investigate replication arrest is to disturb dNTP production and the cellular response to such a disturbance has been thoroughly studied. Here, we use another approach investigating the requirement of protein biosynthesis for functional DNA replication by utilizing the specific protein translation inhibitor cycloheximide. We find that replication is rapidly impaired by inhibition of protein biosynthesis, however, we find no evidence of DNA damage formation. This is surprising and implicate that this response is distinct from arresting DNA replication by inhibition of dNTP production.

Additional information

Funding

This work was supported by the Swedish Children's Cancer Foundation (S.H, N.G), the Swedish Society for Medical Research (N.G), Swedish Research Council, the European Research Council, Göran Gustafsson Foundation, Swedish Cancer Society, the Swedish Pain Relief Foundation, and the Torsten and Ragnar Söderberg Foundation (T.H).