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J. Biol. Chem., Vol. 279, Issue 48, 50472-50481, November 26, 2004

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Residues within the B' Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2^*

Miran Yoon-Robarts, Amanda G. Blouin, Svenja Bleker¶, Jürgen A. Kleinschmidt¶, Aneel K. Aggarwal||, Carlos R. Escalante||**, and R. Michael Linden

From the Departments of Gene and Cell Medicine and Microbiology and the ^||Structural Biology Center, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029 and the ^¶Applied Tumor Virology Program, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.

Received for publication, April 8, 2004 , and in revised form, September 2, 2004.

^* This work was supported by National Institutes of Health Grants R01 GM/AI62234 (to R. M. L.) and R01 AI41706 (to A. K. A.) as well as by Bundesministerium für Forschung und Technik Grant No. 01KV9805/4 (to J. A. K.). 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.

Both authors contributed equally to this work.

^** To whom correspondence may be addressed. E-mail: escalant{at}inka.mssm.edu. To whom correspondence may be addressed: Dept. of Gene and Cell Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Tel.: 212-659-8278; Fax: 212-849-2437; E-mail: Michael.Linden{at}mssm.edu.

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