Cytosolic DNA sensing by cGAS/STING promotes TRPV2-mediated Ca2+ release to protect stressed replication forks.

The protection of DNA replication forks under stress is essential for genome maintenance and cancer suppression. One mechanism of fork protection involves an elevation in intracellular Ca2+ ([Ca2+]i), which in turn activates CaMKK2 and AMPK to prevent uncontrolled fork processing by Exo1. How replication stress triggers [Ca2+]i elevation is unclear. Here, we report a role of cytosolic self-DNA (cytosDNA) and the ion channel TRPV2 in [Ca2+]i induction and fork protection. Replication stress leads to the generation of ssDNA and dsDNA species that, upon translocation into cytoplasm, trigger the activation of the sensor protein cGAS and the production of cGAMP. The subsequent binding of cGAMP to STING causes its dissociation from TRPV2, leading to TRPV2 derepression and Ca2+ release from the ER, which in turn activates the downstream signaling cascade to prevent fork degradation. This Ca2+-dependent genome protection pathway is also activated in response to replication stress caused by oncogene activation.

Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress.

Uncontrolled resection of replication forks under stress can cause genomic instability and influence cancer formation. Extensive fork resection has also been implicated in the chemosensitivity of "BReast CAncer gene" BRCA-deficient cancers. However, how fork resection is controlled in different genetic contexts and how it affects chromosomal stability and cell survival remains incompletely understood. Here, we report a novel function of the transcription repressor ZKSCAN3 in fork protection and chromosomal stability maintenance under replication stress. We show disruption of ZKSCAN3 function causes excessive resection of replication forks by the exonuclease Exo1 and homologous DNA recombination/repair protein Mre11 following fork reversal. Interestingly, in BRCA1-deficient cells, we found ZKSCAN3 actually promotes fork resection upon replication stress. We demonstrate these anti- and pro-resection roles of ZKSCAN3, consisting of a SCAN box, Kruppel-associated box, and zinc finger domain, are mediated by its SCAN box domain and do not require the Kruppel-associated box or zinc finger domains, suggesting that the transcriptional function of ZKSCAN3 is not involved. Furthermore, despite the severe impact on fork structure and chromosomal stability, depletion of ZKSCAN3 did not affect the short-term survival of BRCA1-proficient or BRCA1-deficient cells after treatment with cancer drugs hydroxyurea, PARPi, or cisplatin. Our findings reveal a unique relationship between ZKSCAN3 and BRCA1 in fork protection and add to our understanding of the relationships between replication fork protection, chromosomal instability, and chemosensitivity.