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Originally published In Press as doi:10.1074/jbc.M008829200 on November 16, 2000

J. Biol. Chem., Vol. 276, Issue 11, 7843-7849, March 16, 2001
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In Eukaryotic Flap Endonuclease 1, the C Terminus Is Essential for Substrate Binding*

Manuel Stucki, Zophonías O. JónssonDagger , and Ulrich Hübscher§

From the Institut für Veterinärbiochemie, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.

* This work was supported by Swiss National Science Foundation Grants 31 43.138 95/2 and 31 61361.00 and by the Kanton of Zürich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.

§ To whom correspondence should be addressed. Tel.: 411-635-54-72; Fax: 411-635-68-40; e-mail: hubscher@vetbio.unizh.ch.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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