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J. Biol. Chem., Vol. 278, Issue 36, 34011-34017, September 5, 2003

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A Biochemical Characterization of the Adeno-associated Virus Rep40 Helicase^*

Roy F. Collaco, Vivian Kalman-Maltese, Andrew D. Smith, John David Dignam and James P. Trempe 

From the Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg²⁺ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for K_ATP in the ATPase assay are 1.1 ± 0.2 mM and 1.2 ± 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for V_max are 220 ± 10 and 1,500 ± 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.

Received for publication, February 12, 2003 , and in revised form, June 13, 2003.

^* This work was supported by National Institutes of Health Grants AI51471 and GM64765. 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.

To whom reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Ohio, Block Health Science Bldg., Rm. 408, 3035 Arlington Ave., Toledo, OH 43614. Tel.: 419-383-4103; Fax: 419-383-6228; E-mail: jtrempe{at}mco.edu.

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