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J. Biol. Chem., Vol. 279, Issue 23, 24394-24402, June 4, 2004

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Interaction Interface of Human Flap Endonuclease-1 with Its DNA Substrates^*

Junzhuan Qiu, Ren Liu¶, Brian R. Chapados||, Mark Sherman**, John A. Tainer||, and Binghui Shen¶

From the Departments of Radiation Research and ^**Bio-informatics and the ^¶Graduate Program in Biological Science, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010 and the ^||Department of Molecular Biology and Skaggs Institute of Chemical Biology, the Scripps Research Institute, La Jolla, California 92122

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg⁴⁷, Arg⁷⁰, and Lys³²⁶-Arg³²⁷ of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys²⁴⁴-Arg²⁴⁵ interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg⁷⁰ and Arg⁴⁷ were determined to interact with the sites around the 2nd nucleotide (Arg⁷⁰) or the 5th/6th nucleotide (Arg⁴⁷) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1·DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39–50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.

Received for publication, February 10, 2004 , and in revised form, March 17, 2004.

^* This work was supported by NCI, National Institutes of Health Grants R01CA073764 (to B. S.) and R01CA081967 (to J. A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table S1.

These authors contributed equally to this work.

To whom correspondence should be addressed: Radiation Research, City of Hope National Medical Center, 1500 East Duarte, Duarte, CA 91010. Tel.: 626-301-8879; Fax: 626-301-8280; E-mail: bshen{at}coh.org.

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