Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity

Abstract

The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make 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 believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with 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 activity 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.

Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

“…From previously published studies, Rep proteins with mutations at these positions were not expected to display nicking activity (69). We suspect that the discrepancy between our analysis and others concerning in vitro biochemical activities (69) are related to the MBP-Rep68 fusion protein. With the exception of mutant E504A-68, we did not observe an influence on Rep activity in the context of the Rep68H6 protein.…”

“…3A, lanes 3, 9, and 11, respectively). These results are noteworthy considering that mutants with similar mutations at these positions have previously been shown to be negative for trs endonuclease activity when measured in vitro in the context of the maltose-binding protein (MBP)-Rep68⌬ fusion protein (21,69).…”

“…5). Interestingly, we determined that three of the mutants (Y311F, E465A, and K474,D475A) grouped within this class were active, whereas similar mutants have previously been shown to lack endonuclease activity in the context of the MBP-Rep68 fusion protein (21,69). The discrepancy between these observations suggests a need for concern when evaluating only in vitro Rep activity by using fusion proteins.…”

“…Mutant studies of the Rep proteins have indicated that the activities of Rep can be divided into partially distinct functional domains (Fig. 1A) that are spread throughout the protein (14,21,43,48,(68)(69)(70)(71)77). These include regions required for binding to the ITR, a putative nucleoside triphosphate (NTP) binding/ATPase domain, a nuclear localization domain, and residues putatively required for nicking and helicase functions.…”

“…Rep proteins with mutations within the NTP binding/ ATPase domain that lacked trs endonuclease and viral replication were also defective for transactivation functions, suggesting a need for further mutant analysis (43). Since most mutants disrupt multiple Rep-mediated functions for the AAV life cycle, the detailed characterization of distinct functions has been difficult (21,43,48,(68)(69)(70)(71)77).…”