The archaeal DNA primase: Biochemical characterization of the p41-p46 complex from pyrococcus furiosus

Lidong Liu, Kayoko Komori, Sonoko Ishino, Arnaud A. Bocquier, Isaac K.O. Cann, Daisuke Kohda, Yoshizumi Ishino

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Abstract

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase α-primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [γ-32P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase α-primase complex.

Original languageEnglish (US)
Pages (from-to)45484-45490
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number48
DOIs
StatePublished - Nov 30 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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