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Journal of Virology, April 2000, p. 3122-3129, Vol. 74, No. 7
0022-538X/00/$04.00+0

An Adeno-Associated Virus (AAV) Initiator Protein, Rep78, Catalyzes the Cleavage and Ligation of Single-Stranded AAV ori DNA

Richard H. Smith and Robert M. Kotin*

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

Received 23 September 1999/Accepted 10 January 2000

The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)-Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn2+ or Mg2+) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded cis-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.

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

Journal of Virology, April 2000, p. 3122-3129, Vol. 74, No. 7
0022-538X/00/$04.00+0


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