DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system

The DNA sequence motifs which direct adeno-associated virus type 2 site-spe cific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements compr ise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory p rotein (Rep) separated by a short DNA spacer from a sequence (terminal reso lution site [TRS]) that can serve as a substrate for Rep-mediated nicking a ctivity (R, M, Linden, P, Ward, C, Giraud, E, Winocour, and K, I. Berns, Pr oc. Natl, Acad, Sci, USA 93:11288-11294, 1996; R, M, Linden, E. Winocour, a nd K, I. Berns, Proc. Natl, Acad. Sci, USA 93:7966-7972, 1996), We now repo rt that episomal integration depends upon both the sequence and the positio n of the spacer DNA separating the RES and TRS motifs, The spacer thus cons titutes a third element required for site-specific episomal integration.