Chiorini J A, Wiener S M, Owens R A, Kyöstió S R, Kotin R M, Safer B
Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-1654.
J Virol. 1994 Nov;68(11):7448-57. doi: 10.1128/JVI.68.11.7448-7457.1994.
Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68 delta produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, delta 57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68 delta of approximately 8 x 10(-10) M was determined for the wt ITR and delta 57 ITR probes. Truncation of delta 57 ITR to generate delta 28 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than delta 57 ITR. Extension of delta 28 ITR with nonspecific sequence restored the ability of MBP-Rep68 delta to bind to delta 28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the delta 57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep68 delta. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep68 delta or wt Rep68 to hairpin ITR or delta 57 ITR was indistinguishable. However, the binding activity of MBP-Rep68 delta to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68 delta to the nonhairpin delta 57 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep68 delta binding, its presence does affect the trs cleavage activity of the protein.
2型腺相关病毒回文倒置末端重复序列（ITRs）的复制需要病毒非结构Rep蛋白的多种功能。这些功能包括与ITR结合、在末端分辨率位点（trs）切割双链复制中间体，然后进行链置换并从切口处开始合成。本报告证明了在大肠杆菌中产生的重组融合麦芽糖结合蛋白（MBP）-Rep68 delta和野生型（wt）Rep68与线性截短形式的ITR（delta 57 ITR）结合的能力，其亲和力与wt发夹ITR相似。对于wt ITR和delta 57 ITR探针，测定了MBP-Rep68 delta的解离常数约为8×10^(-10) M。将delta 57 ITR截短以产生delta 28 ITR，其保留了GCTC重复基序但没有trs，其结合效率比delta 57 ITR至少低10倍。用非特异性序列延伸delta 28 ITR恢复了MBP-Rep68 delta与delta 28 ITR结合的能力。因此，高亲和力结合似乎需要侧翼序列以及完整的GCTC重复基序来稳定。用DdeI切割delta 57 ITR探针，其截断了侧翼序列，并且先前已证明可抑制Rep68的结合，这也抑制了MBP-Rep68 delta的结合。通过一系列跨越DNase I足迹实验中MBP-Rep78保护区域的寡核苷酸探针进一步确定了稳定结合的要求。MBP-Rep68 delta或wt Rep68与发夹ITR或delta 57 ITR的结合活性没有区别。然而，MBP-Rep68 delta与DNA的结合活性似乎与trs内切核酸酶活性无关。MBP-Rep68 delta与非发夹delta 57 ITR的切口和共价连接效率比其与含发夹ITR的连接效率低约100倍。因此，尽管ITR的发夹部分似乎在MBP-Rep68 delta结合的识别和稳定中不起作用，但其存在确实会影响该蛋白的trs切割活性。
