INTERACTION OF WILD-TYPE AND MUTANT ADENOASSOCIATED VIRUS (AAV) REP PROTEINS ON AAV HAIRPIN DNA

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AA V) bind to AAV terminal repeat hairpin DNA and can mediate site-specif ic nicking in vitro at the terminal resolution site (trs) within the t erminal repeats. To define the regions of the Rep proteins required fo r these functions, a series of truncated Rep78 derivatives was created . Wild-type and mutant proteins were synthesized by in vitro translati on and analyzed for AAV hairpin DNA binding, trs endonuclease activity , and interaction on hairpin DNA. Amino-terminal deletion mutants whic h lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carb oxyl-terminal region of Rep78 di not eliminate hairpin DNA binding unt il the deletion reached amino acid 443. The electrophoretic mobility o f the Rep-specific protein-DNA complexes ws inversely related to the m olecular weight of the Rep derivative. Analysis of the C-terminal dele tion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary f or DNA binding. When endonuclease-positive, truncated Rep proteins tha t bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA comp lexes was observed. This smear migrated at an intermediate position wi th respect to the bands generated by the proteins individually. An ant ibody recognizing only the full-length protein produced a novel supers hift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep protein s can form hetero-oligomers on the AAV hairpin DNA.