A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2.
We report the characterization of a DNA primase/polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus- and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases. PolpTN2 exhibited primase, polymerase and nucleotidyl transferase activities and specifically incorporates dNTPs, to the exclusion of rNTPs. PolpTN2 could efficiently prime DNA synthesis by the T. nautilus PolB DNA polymerase, suggesting that it is used in vivo as a primase for pTN2 plasmid replication. The N-terminal PriS-like domain of PolpTN2 exhibited all activities of the full-length enzyme but was much less efficient in priming cellular DNA polymerases. Surprisingly, the N-terminal domain possesses reverse transcriptase activity. We speculate that this activity could reflect an ancestral function of AEP proteins in the transition from the RNA to the DNA world.
Institut Pasteur Unité Biologie Moléculaire du Gène chez les Extrêmophiles, 25 rue du Docteur Roux, 75015 Paris, France, CNRS UMR 7138 Systématique, Adaptation, Evolution, Université Paris 6 quai Saint-Bernard, 75252 Paris Cedex 05, France and Univ Paris-Sud Institut de Génétique et Microbiologie, CNRS UMR 8621, Orsay 91406, France.
Sukhvinder
S
Gill
Mart
M
Krupovic
Nicole
N
Desnoues
Pierre
P
Béguin
Guennadi
G
Sezonov
Patrick
P
Forterre
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; C034477:RNA primers; D012313:RNA; D004247:DNA; D019915:DNA Primase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase