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Journal of Virology, March 2000, p. 2936-2942, Vol. 74, No. 6
0022-538X/00/$04.00+0

Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates

Michael D. Davis,dagger Jianwen Wu,Dagger and Roland A. Owens*

Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892

Received 12 October 1999/Accepted 15 December 1999

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722-2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.

* Corresponding author. Mailing address: Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bldg. 8, Rm. 310, 8 Center Dr. MSC 0840, Bethesda, MD 20892-0840. Phone: (301) 496-3359. Fax: (301) 402-0053. E-mail: ro6n{at}nih.gov.

dagger Present address: Department of Health Administration, City of Detroit, Detroit, MI 48202.

Dagger Present address: Department of Medicine, Louisiana State University School of Medicine, New Orleans, LA 70112.

Journal of Virology, March 2000, p. 2936-2942, Vol. 74, No. 6
0022-538X/00/$04.00+0


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