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J. Biol. Chem., Vol. 278, Issue 7, 4912-4918, February 14, 2003

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Identification of Active Site Residues of the Adeno-associated Virus Type 2 Rep Endonuclease^*

Miran Yoon-Robarts^§ and R. Michael Linden^¶

From the  Carl C. Icahn Institute for Gene Therapy and Molecular Medicine and the ^¶ Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.

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