title: Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.
abstract: We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small 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 synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), 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 been shown to be essential for catalysis in the mouse primase. Of the four cysteine 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 report on the biochemical characterisation of an archaeal DNA primase.
authors.affiliation: Imperial College of Science, Technology, and Medicine, Biophysics Section, Blackett Laboratory, London SW7 2BZ, UK.
authors.forename: G
authors.initials: G
authors.lastname: Desogus
authors.forename: S
authors.initials: S
authors.lastname: Onesti
authors.forename: P
authors.initials: P
authors.lastname: Brick
authors.forename: M
authors.initials: M
authors.lastname: Rossi
authors.forename: F M
authors.initials: FM
authors.lastname: Pisani
mesh_terms: D000595:Amino Acid Sequence; D019915:DNA Primase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D019915:DNA Primase