ASYMMETRIC REPLICATION IN-VITRO FROM A HUMAN SEQUENCE ELEMENT IS DEPENDENT ON ADENOASSOCIATED VIRUS REP PROTEIN

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integr ates preferentially into a defined region of human chromosome 19. Sout hern blots of genomic DNA from latently infected cell lines revealed t hat the provirus was not simply inserted into the cellular DNA. Both, the proviral and adjoining cellular DNA organization indicated that in tegration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for target ed integration has remained obscure. The two larger nonstructural prot eins (Rep68 and Rep78) of AAV bind to a sequence element that is prese nt in both the integration locus (P1) and the AAV inverted terminal re peat. This binding may be important for targeted integration. To inves tigate the mechanism of targeted integration, we tested the cloned int egration site subfragment in a cell-free replication assay in the pres ence or absence of recombinant Rep proteins. Extensive, asymmetric rep lication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted term inal repeat. Replication apparently initiates from a 3'-OH generated b y the sequence specific nicking activity of Rep. This results in a cov alent attachment between Rep and the 5'-thymidine of the nick The comp lexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.