| Abstrakt: |
The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE'), and the trs. Previously, we determined the sequences at the trs required for Rep-mediated cleavage and demonstrated that the trs endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at the trs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE' during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation during trs nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE' and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trs greatly inhibit Rep nicking. Substitutions within the RBE' also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE' during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trs cleavage steps of the endonuclease reaction. On the other hand, RBE' contacts seem to be required primarily for TR unwinding and formation of the trs stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE', and the trs. |
