Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells

Background: The cGAS/STING pathway, part of the innate immune response to foreign DNA, can be activated by cell’s own DNA arising from the processing of the genome, including the degradation of nascent DNA at arrested replication forks, which can be upregulated in cancer cells. Recent evidence raises a possibility that the cGAS/STING pathway may also modulate the very processes that trigger it, e.g., DNA damage repair or processing of stalled forks. Methods: We manipulated STING levels in human cells by depleting or re-expressing it, and assessed the effects of STING on replication using microfluidics-assisted replication track analysis, or maRTA, a DNA fiber assay, as well as immuno-precipitation of nascent DNA, or iPOND. We also assessed STING subcellular distribution and its ability to activate. Results: Depletion of STING suppressed and its re-expression in STING-deficient cancer cells upregulated the degradation of nascent DNA at arrested replication forks. Replication fork arrest was accompanied by the STING pathway activation, and a STING mutant that does not activate the pathway failed to upregulate nascent DNA degradation. cGAS was required for STING’s effect on degradation, but this requirement could be bypassed by treating cells with a STING agonist. Cells expressing inactive STING had a reduced level of RPA on parental and nascent DNA of arrested forks and a reduced CHK1 activation compared to cells with the wild type STING. STING also affected unperturbed fork progression in a subset of cell lines. STING fractionated to the nuclear fractions enriched for structural components of chromatin and nuclear envelope, and furthermore, it associated with the chromatin of arrested replication forks as well as post-replicative chromatin. Conclusion: Our data highlight STING as a determinant of stalled replication fork integrity, thus revealing a novel connection between the replication stress and innate immune responses.