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J. Biol. Chem., Vol. 283, Issue 3, 1732-1743, January 18, 2008

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The Iron-containing Domain Is Essential in Rad3 Helicases for Coupling of ATP Hydrolysis to DNA Translocation and for Targeting the Helicase to the Single-stranded DNA-Double-stranded DNA Junction^*

Robert A. Pugh, Masayoshi Honda, Haley Leesley, Alvin Thomas^¶, Yuyen Lin, Mark J. Nilges^||, Isaac K. O. Cann^¶**, and Maria Spies¹

From the Department of Biochemistry, Department of Animal Sciences, and ^¶Department of Microbiology, ^||Illinois EPR Research Center, and ^**Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.

Received for publication, August 22, 2007 , and in revised form, November 8, 2007.

^* This work was supported by the University of Illinois start-up funds (to M. S.) and by National Science Foundation Grant MCB-0238451 (to I. K. O. C.). 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 supplemental Methods, additional references, and Figs. S1-S6.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, 493 Roger Adams Laboratory/MC-712, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801-3602. Tel.: 217-244-9493; Fax: 217-244-5858; E-mail: mspies{at}life.uiuc.edu.