Fig 2: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P < 0.01 (unpaired t test). Data are representative of three experiments with similar results

Fig 3: Multiple mechanisms are involved in inhibition of cGAS-mediated pathways in mitotic cells.a, b CDK1 inhibition partially restores cGAMP production. Asynchronized (Asyn) or synchronized (Mitotic) HT1080 cells were left untreated or treated with RO-3306 (10 μM) for 30 min, and then cell extracts containing cGAMP were delivered to digitonin-permeabilized Raw264.7 cells for 4 h before qPCR analysis for mRNA levels of the indicated genes (a), or were directly analyzed by mass spectrometry (b). **P < 0.01, ***P < 0.001 (Student’s t-test, unpaired, two-tailed). Data shown are mean ± S.D. of one representative experiment performed in triplet (a). c Effects of CDK1 inhibition on activation of downstream components of cGAS in mitotic cells. Synchronized (Mitotic) HT1080 cells were left untreated or treated with RO-3306 (10 μM) for the indicated times before immunoblotting analysis with the indicated antibodies. Asynchronized (Asyn) HT1080 cells transfected with HT-DNA were used as a positive control. d Inhibition of downstream components of cGAS in mitosis. HT1080 cells asynchronized (Asyn) or synchronized by paclitaxel (Mitotic) were left mock-transfected or transfected with cGAMP for the indicated times before immunoblotting analysis with the indicated antibodies. e cGAMP-induced recruitment of TBK1 to MITA is barely affected in mitotic cells. Asynchronized (Asyn) or synchronized (Mitotic) HT1080 cells were left untreated or treated with cGAMP for 2 h followed by co-immunoprecipitation and immunoblotting analysis. f CA but not OA restores mcGas phosphorylation at S291. Raw264.7 cells were synchronized by nocodazole, pretreated with the phosphatase inhibitors (100 nM) for 30 min, followed by RO-3306 treatment (10 μM) for 30 min or release into fresh medium for 2 h before immunoblotting analysis. g CDK1 and PP1 coordinately regulate cGAS phosphorylation during mitosis. Synchronized (Mitotic) Raw264.7 cells were pretreated with RO-3306 (10 μM) for 30 min then treated with CA or OA (100 nM) for 30 min, or were pretreated with CA or OA (100 nM) for 30 min and then treated with RO-3306 (10 μM) for 30 min. The cells were then analyzed by immunoblots with the indicated antibodies. Data shown are representative of three (a) or two (d–g) biological repeats.

Fig 4: ZCCHC3 is important for cytosolic dsDNA-induced oligomerization of cGAS. a Effects of ZCCHC3 on self-association of cGAS. HEK293 cells were transfected with the indicated plasmids for 20 h. The cells were then left un-treated or treated with HSV120 for 4 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. b Effects of ZCCHC3-deficiency on self-association of cGAS. ZCCHC3-KO and control HEK293 cells were transfected with the indicated plasmids for 20 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c Effects of ZCCHC3-deficiency on oligomerization of cGAS induced by transfected VACV70. Zcchc3+/+ and Zcchc3−/− MLFs were left un-treated or treated with VACV70 for 4 h. Cell lysates were then fractionated by SDD-AGE and SDS-PAGE and analyzed by immunoblots with the indicated antibodies. Data are representative of three experiments with similar results

Fig 5: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a. c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3+/+ and Zcchc3−/− MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3−/−MLFs. Zcchc3−/− MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas+/+ and cGas−/− L929 cells. cGas+/+ and cGas−/− L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P < 0.05, **P < 0.01 (unpaired t test). Data are representative of at least two experiments with similar results (mean ± SD, n = 3 independent samples in e, f)