FIG. 2.

Rep68 mediates AAV-AAVS1 junction formation specifically at the RBS in vitro. (A) Schematic diagrams of the AAV genome and the plasmid pBSAAVS1 containing a 1.6-kb AAVS1 sequence (left) and primer sets (right) used for in vitro junction formation assays. The positions of the primers used are indicated in the diagrams. (B) rRep68 used in this study was visualized by silver staining. The positions of molecular mass markers are shown on the right. (C) In vitro junction formation assays were performed with or without 1.5 pmol of rRep68. The recombination products and the indicated amounts of PCR standards were subjected to PCR analysis with various primer sets as shown in panel A. The PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The positions of size markers are shown on the right. (D) Junction formation assays were performed with the indicated amounts of rRep68 and wild-type (w) or mutant (mt) pBSAAVS1. The recombination products were examined by PCR with primer set 1 (A). The positions of size markers are shown on the right. Quantitation of the results from three independent experiments is shown below. Data represent the means ± standard errors of the mean. (E) AAV-AAVS1 junction sequences from eight clones. The RBS-flanking regions of the two substrates are shown above. In the junction sequences, overlapping sequences between AAV and AAVS1 are indicated by open boxes, and respective junction points are indicated by numbers according to the published numbering system (16). The RBS sequences are indicated by letters. nt, nucleotide.