Distinct Concentration-dependent dsDNA-binding Modes of a Dinoflagellate Cold Shock Domain Protein Provide Insight...

Coral reef ecosystems depend on symbiotic dinoflagellates, yet the molecular mechanisms that regulate their growth remain poorly understood. Dinoflagellates possess unusually large genomes but remarkably few transcription factors, most of which contain a cold shock domain (CSD). In this study, we investigated transcriptional responses associated with growth stimulation in the symbiotic dinoflagellate Breviolum minutum. Exposure to zinc oxide nanoparticles (ZnO NPs) significantly promoted cell proliferation, and transcriptome analysis identified a CSD protein gene, designated BmCSP1, as one of the few upregulated genes encoding transcription-related proteins under this growth-promoting condition. Phylogenetic analysis revealed that BmCSP1 belongs to a highly divergent dinoflagellate CSD clade. To examine its biochemical properties, we analyzed the recombinant CSD of BmCSP1 and compared it with Escherichia coli CspA. BmCSP1 bound single-stranded DNA and RNA with apparent affinities similar to those of EcCspA but exhibited stronger apparent binding to double-stranded DNA (dsDNA). Electrophoretic mobility shift assays further revealed a distinctive concentration-dependent dsDNA-binding behavior: at lower concentrations, BmCSP1 formed sharp, discrete protein-DNA complexes, whereas at higher concentrations the electrophoretic behavior of the complexes became increasingly heterogeneous. Comparative analyses using supercoiled, open circular, and linearized plasmid DNA further demonstrated that BmCSP1-dsDNA interactions are strongly influenced by DNA topology. In addition, both BmCSP1- and EcCspA-dsDNA complexes remained stable after heating to 95 °C, despite loss of dsDNA-binding activity of the free proteins above 55 °C. These findings identify BmCSP1 as a dinoflagellate CSD protein with unique topology-dependent dsDNA-binding modes and exceptional complex stability, providing new insight into how a limited repertoire of transcription factors may contribute to genome organization and transcriptional regulation in dinoflagellates.