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Journal of Virology, November 1999, p. 9325-9336, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rep-Mediated Nicking of the Adeno-Associated Virus Origin Requires Two Biochemical Activities, DNA Helicase Activity and Transesterification

J. Rodney Brister and Nicholas Muzyczka*

Department of Molecular Genetics and Microbiology and University of Florida Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida 32610

Received 8 June 1999/Accepted 11 August 1999

The single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trs sequence necessary for Rep cleavage, 3'-CCGGT/TG-5', and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at the trs. Interestingly, Rep nicking on a TR substrate that fixes this trs stem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trs and extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, P.O. Box 100266 JHMHSC, Gainesville, FL 32610. Phone: (352) 392-8541. Fax: (352) 392-5914. E-mail: muzyczka{at}college.med.ufl.edu.

Journal of Virology, November 1999, p. 9325-9336, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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