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J. Biol. Chem., Vol. 277, Issue 40, 37840-37847, October 4, 2002

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Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii^*

Eriko Matsui, Krishnasastry V. Musti^§, Junko Abe, Kazuhiko Yamasaki^¶, Ikuo Matsui, and Kazuaki Harata

From the  Biological Information Research Center and the ^¶ Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki 305-566, Japan

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 Å. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg⁴⁰ and Arg⁴² on small loop 1, a cluster Lys¹⁹³-Lys¹⁹⁵ on small loop 2, and two sites, Arg⁹⁴ and Arg¹¹⁸-Lys¹¹⁹, on the large loop were identified as binding sites. Lys⁸⁷ on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg⁹⁴ and Lys¹⁹³-Lys¹⁹⁵ located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg¹¹⁸-Lys¹¹⁹ was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

The atomic coordinates and the structure factors (code 1MC8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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