title: The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair?
abstract: We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase alpha-primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.
authors.affiliation: Laboratoire de Microbiologie des Environnements Extrêmes, UMR6197, Ifremer, BP 70, F-29280 Plouzané, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Le Breton
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Henneke
authors.forename: Cédric
authors.initials: C
authors.lastname: Norais
authors.forename: Didier
authors.initials: D
authors.lastname: Flament
authors.forename: Hannu
authors.initials: H
authors.lastname: Myllykallio
authors.forename: Joël
authors.initials: J
authors.lastname: Querellou
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Raffin
mesh_terms: D003001:Cloning, Molecular; D019915:DNA Primase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D047468:Immunoprecipitation; D007700:Kinetics; D043903:Pyrococcus abyssi / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D019915:DNA Primase