Formation of dAMP-glycerol and dAMP-Tris Derivatives by Thermococcus kodakaraensis DNA Primase*

Background: Archaeal primase catalyzed the DNA-independent synthesis of oligonucleotide-like products from dNTPs. Results: Mass spectrometry, NMR, and biochemical analyses identified these products as dNMP-glycerol and dNMP-Tris derivatives. Conclusion: Archaeal primase catalyzes the formation of phosphodiester bonds between dNMPs and various acceptors. Significance: Archaeal primase forms DNA-independent covalent adducts between deoxynucleotides and acceptors that mimic de novo aborted initiation reactions. In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.