Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of ade no-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 req uired for endonuclease activity. We demonstrate that several mutant protein s which are endonuclease negative on a fully duplex hairpin substrate are e ndonuclease positive on a partially single-stranded hairpin substrate. Trun cation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is be lieved to involve the covalent attachment of Rep68/78 to the trs via a phos phate-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 t he first 200 amino acids of the Rep68 moiety of a maltose binding protein-R ep68/78 fusion protein to phenylalanine, Only mutation of tyrosine 156 resu lted in a protein incapable of covalent attachment to a partially single-st randed hairpin substrate, suggesting that tyrosine 156 is part of the endon uclease active site.