Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus

Kayoko Komori, Shinzi Sakae, Ryosuke Fujikane, Kosuke Morikawa, Hideo Shinagawa, Yoshizumi Ishino

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg2+ at 5-10 mM was optimal. Mn2+ could substitute for Mg2+, but it was much less efficient than Mg2+ as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCI at ~200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of Sulfolobus, the Holliday junction resolving activity detected in P.furiosus cell extract thus far is only derived from Hjc.

Original languageEnglish
Pages (from-to)4544-4551
Number of pages8
JournalNucleic acids research
Volume28
Issue number22
Publication statusPublished - Nov 15 2000
Externally publishedYes

Fingerprint

Holliday Junction Resolvases
Pyrococcus furiosus
Cruciform DNA
Sulfolobus
Endonucleases
Homologous Recombination
DNA
Archaea
Divalent Cations
Cell Extracts
Amino Acid Sequence
Hot Temperature
Escherichia coli
Proteins

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Komori, K., Sakae, S., Fujikane, R., Morikawa, K., Shinagawa, H., & Ishino, Y. (2000). Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. Nucleic acids research, 28(22), 4544-4551.

Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. / Komori, Kayoko; Sakae, Shinzi; Fujikane, Ryosuke; Morikawa, Kosuke; Shinagawa, Hideo; Ishino, Yoshizumi.

In: Nucleic acids research, Vol. 28, No. 22, 15.11.2000, p. 4544-4551.

Research output: Contribution to journalArticle

Komori, K, Sakae, S, Fujikane, R, Morikawa, K, Shinagawa, H & Ishino, Y 2000, 'Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus', Nucleic acids research, vol. 28, no. 22, pp. 4544-4551.
Komori K, Sakae S, Fujikane R, Morikawa K, Shinagawa H, Ishino Y. Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. Nucleic acids research. 2000 Nov 15;28(22):4544-4551.
Komori, Kayoko ; Sakae, Shinzi ; Fujikane, Ryosuke ; Morikawa, Kosuke ; Shinagawa, Hideo ; Ishino, Yoshizumi. / Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. In: Nucleic acids research. 2000 ; Vol. 28, No. 22. pp. 4544-4551.
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