JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M103397200 on July 26, 2001

J. Biol. Chem., Vol. 276, Issue 39, 36295-36302, September 28, 2001
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Stimulation of Eukaryotic Flap Endonuclease-1 Activities by Proliferating Cell Nuclear Antigen (PCNA) Is Independent of Its in Vitro Interaction via a Consensus PCNA Binding Region*

Geoffrey FrankDagger , Junzhuan QiuDagger , Li Zheng, and Binghui Shen§

From the Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California 91010

Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q1X2X3(L/I)4X5X6F7(F/Y)8, although our results fail to implicate Q1 (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.

* This work was supported by National Institutes of Health Grant CA73764 (to B. H. S.).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 These authors contributed to this work equally.

§ To whom correspondence should be addressed: Dept. of Cell and Tumor Biology, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010. Tel.: 626-301-8879; Fax: 626-301-8972; E-mail: bshen@coh.org.

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


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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.