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J. Biol. Chem., Vol. 282, Issue 43, 31713-31724, October 26, 2007

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Crystal Structure of Bacteriophage T4 5' Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates^*

Juliette M. Devos, Stephen J. Tomanicek¹, Charles E. Jones, Nancy G. Nossal, and Timothy C. Mueser²

From the Department of Chemistry, The University of Toledo, Toledo, Ohio, 43606 and Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR [PDB] ) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.

Received for publication, April 16, 2007 , and in revised form, August 6, 2007.

The atomic coordinates and structure factors (code 2IHN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Science Foundation CAREER Award 0346960 (to T. C. M.) and by the Intramural Research Program of the NIDDK, National Institutes of Health (to N. G. N.). 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 two movies.

¹ Supported in part by American Heart Association Ohio Valley Affiliate Pre-doctoral Fellowship Award 0315174B.

We dedicate this publication to Dr. Nancy G. Nossal, who passed away after the completion of this work and the preparation of this manuscript. Her involvement and valuable suggestions greatly contributed to the quality of this research.

² To whom correspondence should be addressed: Dept. of Chemistry, University of Toledo, M.S. 602, 2801 W. Bancroft St., Toledo, OH 43606. Tel.: 419-530-1510; Fax: 419-530-4033; E-mail: timothy.mueser{at}utoledo.edu.