MUTATIONAL ANALYSIS OF THE ADENOASSOCIATED VIRUS REP68 PROTEIN - IDENTIFICATION OF CRITICAL RESIDUES NECESSARY FOR SITE-SPECIFIC ENDONUCLEASE ACTIVITY

The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) ar e multifunctional proteins which contain overlapping amino acid sequen ces. They are required for viral replication and preferential integrat ion of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins ma ke a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is beli eved to involve the covalent attachment of Rep68 or Rep78 at the cut s ite via a phosphotyrosine linkage. In an attempt to identify the activ e-site tyrosine residue of Rep78 and Rep68, tyrosine residues were sit e specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protei n-Rep68 fusion (MBP-Rep68 Delta) expression vector. The mutant MBP-Rep 68 Delta proteins were expressed in Escherichia coli cells, purified w ith amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity , only the mutation of tyrosine 152 abrogated the endonuclease activit y with no discernible effect on the helicase or DNA-binding activities . Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.