Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The a nalysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the sma ll subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthe sise oligoribonucleotides on various pyrimidine single-stranded DNA templat es [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg 2+, Mn2+ or Zn2+, and is additionally stimulated by the monovalent cation K +. A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have b een shown to be essential for catalysis in the mouse primase. Of the four c ysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first re port on the biochemical characterisation of an archaeal DNA primase.