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J Virol, June 1998, p. 4874-4881, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Rep52 Gene Product of Adeno-Associated Virus Is a DNA Helicase with 3'-to-5' Polarity

Richard H. Smith and Robert M. Kotin*

Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892

Received 21 November 1997/Accepted 26 February 1998

The rep gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P5 and P19. The P5-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P19-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3'-to-5' DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a trans-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of trans dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.

* Corresponding author. Mailing address: Molecular Hematology Branch, NHLBI, Bldg. 10, Rm. 7D18, 10 Center Dr., MSC 1654, Bethesda, MD 20892-1654. Phone: (301) 496-1594. Fax: (301) 496-9985. E-mail: kotinr{at}fido.nhlbi.nih.gov.

J Virol, June 1998, p. 4874-4881, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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