Selective cleavage of AAVS1 substrates by the adeno-associated virus type 2 Rep68 protein is dependent on topological and sequence constraints

The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are requ ired for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 i nitiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (hs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nick ing RES and trs homologues present in AAVS1. However, Rep78 and Rep68 cleav e in vitro AAVS1 duplex-linear substrates much less efficiently than hairpi nned ITRs. In this study, Ive show that the AAV-2 Rep68 endonuclease activi ty is affected by the topology of the substrates in that it efficiently cle aves in vitro in a site- and strand-specific manner the AAVS1 trs only if t his sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesi s in the context of SC templates allowed us to elucidate for the first time the AAVS1 trs sequence and position requirements for Rep68-mediated cleava ge. Interestingly, Rep68 did not cleave SC templates containing RES from ot her sites of the human genome. These findings have intriguing implications for AAV 2 site-specific integration in vivo.