Brister J R, Muzyczka N
Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
J Virol. 2000 Sep;74(17):7762-71. doi: 10.1128/jvi.74.17.7762-7771.2000.
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.
单链2型腺相关病毒（AAV）基因组两侧是末端重复序列（TRs），这些序列自身回折形成发夹结构。在AAV DNA复制过程中，TRs在末端切割位点（trs）被病毒编码的Rep蛋白切割。这种起始功能显然需要三个序列元件，即Rep结合元件（RBE）、一个小回文序列（它构成TR内一个内部发夹的单个末端，即RBE'）和trs。此前，我们确定了Rep介导切割所需的trs序列，并证明trs内切酶反应分两个离散步骤进行。第一步，Rep DNA解旋酶活性解开TR，从而在trs处挤出一个茎环结构。第二步，Rep转酯酶活性切割trs。在此，我们研究RBE和RBE'在此过程中的作用。我们的数据表明，在trs切割过程中，Rep以特定方向与RBE相连。这种方向似乎使Rep在AAV TR上排列，允许与RBE'进行特定的核苷酸接触，并将切割导向trs。因此，RBE相对于trs的极性或位置改变会极大地抑制Rep切割。RBE'内的取代也会降低Rep的比活性，但程度较小。有趣的是，Rep在切割过程中与RBE和RBE'的相互作用在功能上似乎是不同的。Rep与RBE的接触对于内切酶反应的DNA解旋酶和trs切割步骤似乎都是必需的。另一方面，RBE'的接触似乎主要是TR解旋和trs茎环结构形成所必需的，而不是切割所必需的。总之，这些结果提出了一个模型，即在起始切割过程中，Rep通过由RBE、RBE'和trs组成的三方切割信号与AAV TR相互作用。
