To clone and characterize Hypsibius henanensis GATA binding protein 3 (HhGATA3) and to investigate its function in radiation tolerance.

The coding sequence of HhGATA3 was amplified from H.henanensis cDNA by PCR, and the protein was analyzed using bioinformatics tools. The subcellular aggregation of HhGATA3 was observed by immunofluorescence. The radioprotective capacity was evaluated via alkaline comet assay and phosphorylated histone H2AX (γH2AX) foci formation in human cells expressing HhGATA3. The knockdown efficiency of small interfering RNA (siRNA) was verified by reverse transcription real-time quantitative PCR (qPCR) in H.henanensis, and the post-irradiation survival rate was measured.

The HhGATA3 gene was cloned. HhGATA3 protein formed phase-separated droplets within cells. Comet and γH2AX foci assays suggested that HeLa cells expressing HhGATA3 had their DNA damage significantly mitigated after X-ray irradiation (P<0.0001). Knockdown of HhGATA3 in H. henanensis markedly decreased the post-irradiation survival rate to approximately 40% (P<0.001).

HhGATA3 plays a critical role in the radiation tolerance of H.henanensis. In human HeLa cells, HhGATA3 can undergo phase separation and mitigate radiation-induced DNA damage.