THE RECOMBINATION SIGNALS FOR ADENOASSOCIATED VIRUS SITE-SPECIFIC INTEGRATION

The adeno-associated virus (AAV) genome integrates site specifically i nto a defined region of human chromosome 19 (termed AAVS1). Using a fu nctional assay for AAV integration into AAVS1 DNA propagated as an epi some, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence con tains the minimum signal required for targeted integration. The recomb ination signal comprises a DNA-binding motif for the AAV regulatory Re p protein [Rep binding site (RBS)] separated by an eight-nucleotide sp acer from a sequence that can act as a substrate for Rep endonucleolyt ic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since bo th the RBS and TRS elements are present in the viral origin of replica tion and are required for AAV replication, targeted integration into c hromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is resp onsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to he required for AAV episomal integration, despite its location adjacent to the recombination signal.