Sphingosine Inhibits the Synthesis of RNA Primers by Primase in Vitro

We have previously shown the presence of sphingomyelin and sphingomyelinase in cell nuclei, suggesting that they may play a role in the intranuclear production of sphingosine, a potent bioactive molecule modulating diverse cellular functions. In the present study, the direct effects of sphingosine (Ci8:1) on the activity of DNA replication/repair polymerases were studied in vitro. Sphingosine had no effect on DNA polymerases a and β and slightly inhibited DNA polymerases γ, δ, and ϵ. In contrast, sphingosine strongly inhibited the activity of primase in a dose-dependent manner. On the other hand, dihydrosphingosine (Cisio), glycolipids, sphingomyelin, and ceramide had no effect on primase activity. Sphingosine equally inhibited the activity of primase complexed with DNA polymerase a, as well as its free form, with a K_i value of 4 µM. A gel-retardation analysis showed that the binding of primase with ³²P-labeled template DNA was suppressed by sphingosine. Inhibition by sphingosine was competitive with the DNA template, but not with the substrate NTPs. After product analysis, a dose-dependent decrease in the amount of RNA primer products, consisting mainly of 10- and 11-mers, was observed in the presence of sphingosine, indicating that it inhibits the synthesis of RNA primers by primase. Sphingosine, however, had no effect on T7 RNA polymerase.