Biochemical re-evaluation of ZCCHC3 reveals single-stranded DNA-selective binding and suppressive modulation of cGAS activity

Cyclic GMP–AMP synthase (cGAS) is a central cytosolic DNA sensor that initiates innate immune signaling through the production of the second messenger 2′,3′-cGAMP. A previous study using a cell-based system suggested that zinc finger CCHC domain‑containing protein 3 (ZCCHC3) functions as a co-sensor for double-stranded DNA (dsDNA), thereby promoting cGAS activation. However, whether ZCCHC3 directly modulates the enzymatic activity of cGAS has remained unclear. Here, we systematically examined the biochemical function of ZCCHC3 using purified in vitro reconstitution systems. Quantitative analysis of cGAS-mediated 2′,3′-cGAMP production revealed that ZCCHC3 does not enhance cGAS catalytic activity in the presence of dsDNA but instead suppresses it in a concentration-dependent manner. Further quantitative analysis revealed that this inhibitory effect was more pronounced than that observed for other nucleic acid–binding proteins. Notably, ZCCHC3 exhibited minimal binding to canonical dsDNA and instead recognized single-stranded DNA. Together, these findings suggest that ZCCHC3 functions as a direct suppressive modulator of cGAS activity, rather than an obligate co-sensor, and suggest that its regulatory function may be linked to recognition of single-stranded DNA.