Enzymatic Characteristics of Thermostable Flap Endonuclease from Pyrococcus horikoshii

　

Eriko MATSUI^*a,c, Satoko KAWASAKI^a,d, Hiroyasu ISHIDA^a, Kazuhiko ISHIKAWA^a, Yoshitsugu KOSUGI^a, Feng-Wen CUI^a, Yutaka KAWARABAYASHI^{a, b}, Ikuo MATSUI^a

　

^a National Institute of Bioscience and Human-technology, 1-1 Higashi, Tsukuba 305, Japan

^b National Institute of Technology and Evaluation, Ministry of International Trade and Industry, Nishihara, Shibuyaku, Tokyo, Japan

^c Japan Tabacco, Aoba-ku, Yokohama, 227 Kanagawa, Japan

^d New Energy and Industrial Technology Development Organization, Higashi ikebukuro, Toshimaku,Tokyo, Japan

　

The hyperthermophilic archaeon Pyrococcus horikoshii lives in marine and terrestrial volcanic environments, which appears to be similar to ancient earth. The P. horikoshii can grow up to 102℃, and the optimal temperature is 95℃. It was suggested that P. furiosus is highly resistant to γradiation and might have a special system to remove the damage DNA. However it is unclear how replication and repair systems function in Archaea.

Recently, a structure-specific endonuclease was purified and characterized in murine and yeast. This enzyme is called a flap endonuclease-1 (FEN-1), and suggested to have a role in replication and repair in eucaryotes. However, there are no reports of FEN activity in Archaea.

To investigate the repair system of P. horikoshii, the FEN homolog gene identified in the P. horikoshii genome was cloned, then overexpressed in E. coli, and the recombinant protein was characterized.

The gene is 1032 bp (343 amino acids) in length. The deduced amino acid sequence has high similarity to the sequence of the mammalian flap endonuclease (37% overall identity). Gel filtration analysis indicated that this protein was estimated a 41 kDa monomer. The enzyme had a flap endonuclease and 5'-3'exonuclease activity. The optimal pH for the flap endonuclease activity peaked between 6.0 and 7.0. The activity was inhibited by high salt concentration. The enzyme was hyperthermostable (Tm=102℃) and the half life was 2 hr at 95℃.

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