McCarty D M, Pereira D J, Zolotukhin I, Zhou X, Ryan J H, Muzyczka N
Department of Microbiology, School of Medicine, University at Stony Brook, New York 11794.
J Virol. 1994 Aug;68(8):4988-97. doi: 10.1128/JVI.68.8.4988-4997.1994.
We have used baculovirus-expressed Rep68 that has been purified to homogeneity to reexamine the binding properties of the Rep protein. We find that Rep68 is capable of binding to a linear DNA sequence that is contained within a 25-bp sequence of the A stem of the adeno-associated virus (AAV) terminal repeat proximal to the B and C palindromes. This has been shown conclusively by demonstrating that Rep68 could specifically bind to a synthetic oligonucleotide containing the 25-bp region in the absence of the other sequences within the terminal repeat. Rep78 was also capable of binding the A stem recognition element, as demonstrated by the fact that a DNA affinity column containing the 25-bp sequence can be used to purify Rep78. The ability to recognize the linear DNA sequence within the A stem provides a mechanism by which the Rep protein can be oriented on the terminal repeat so that only the correct strand is cut at the terminal resolution site (trs site) during terminal resolution. In addition, computer analysis suggests that sequences similar to the A stem element are present within the three AAV promoter regions. Electrophoretic mobility shift experiments clearly demonstrate that the p5 promoter contains a Rep binding sequence. DNase protection experiments indicate that the Rep binding sequence within the p5 promoter is located between the YY1 initiator sequence and the TATA binding site. This position immediately suggests a mechanism by which the Rep protein could act as a repressor or a transactivator of p5 transcription by interacting with either YY1 or TBP. In addition, gel shift experiments suggest that the p19 promoter also contains a Rep binding site. The presence of Rep binding sites upstream of both promoters suggests that these sites may be involved in coordinate regulation of AAV transcription. In addition, we have identified a heterologous Rep binding sequence within pBR322 DNA. A comparison of the sequences within the A stem, p5, and pBR322 binding sites suggests that a repeating GAGC motif is at least part of the Rep recognition sequence. In the accompanying report (D. M. McCarty, J. H. Ryan, S. Zolutukhin, X. Zhou, and N. Muzyczka, J. Virol. 68:4998-5006, 1994), we examine the relative affinity of Rep to the A stem site and the complete terminal repeat. Finally, we also have reexamined the ability of Rep68 and Rep78 to cut at the trs site in substrates that do not contain the B and C palindromes or any apparent secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)
我们使用了经纯化至同质的杆状病毒表达的Rep68，以重新研究Rep蛋白的结合特性。我们发现Rep68能够结合腺相关病毒（AAV）末端重复序列靠近B和C回文序列的A茎25碱基对序列中的一段线性DNA序列。通过证明Rep68在没有末端重复序列中的其他序列时能够特异性结合包含25碱基对区域的合成寡核苷酸，这一点已得到确凿证明。Rep78也能够结合A茎识别元件，这一事实表明含有25碱基对序列的DNA亲和柱可用于纯化Rep78。识别A茎内线性DNA序列的能力提供了一种机制，通过该机制Rep蛋白可在末端重复序列上定向，从而在末端分辨率过程中仅在末端分辨率位点（trs位点）切割正确的链。此外，计算机分析表明，三个AAV启动子区域内存在与A茎元件相似的序列。电泳迁移率变动实验清楚地表明p5启动子含有一个Rep结合序列。DNase保护实验表明p5启动子内的Rep结合序列位于YY1起始序列和TATA结合位点之间。这个位置立即提示了一种机制，通过该机制Rep蛋白可通过与YY1或TBP相互作用而作为p5转录的阻遏物或反式激活因子发挥作用。此外，凝胶迁移实验表明p19启动子也含有一个Rep结合位点。两个启动子上游均存在Rep结合位点，这表明这些位点可能参与AAV转录的协调调控。此外，我们在pBR322 DNA中鉴定出一个异源Rep结合序列。对A茎、p5和pBR322结合位点内序列的比较表明，重复的GAGC基序至少是Rep识别序列的一部分。在随附的报告（D.M.麦卡蒂、J.H.瑞安、S.佐卢图欣、X.周和N.穆齐茨卡，《病毒学杂志》68:4998 - 5006，1994）中，我们研究了Rep对A茎位点和完整末端重复序列的相对亲和力。最后，我们还重新研究了Rep68和Rep78在不包含B和C回文序列或任何明显二级结构的底物中在trs位点切割的能力。（摘要截短于400字）
