IDENTIFICATION OF LINEAR DNA-SEQUENCES THAT SPECIFICALLY BIND THE ADENOASSOCIATED VIRUS REP PROTEIN
IDENTIFICATION OF LINEAR DNA-SEQUENCES THAT SPECIFICALLY BIND THE ADENOASSOCIATED VIRUS REP PROTEIN
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DOI：
10.1128/jvi.68.8.4988-4997.1994
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发表时间：
1994-08-01
影响因子：
5.4
通讯作者：
MUZYCZKA, N
中科院分区：
文献类型：
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作者：
MCCARTY, DM;PEREIRA, DJ;MUZYCZKA, N
我们使用杆状病毒表达的Rep68，该Rep68已被纯化至均一，以重新检测Rep蛋白的结合特性。我们发现Rep68能够与线性DNA序列结合，该序列包含在腺相关病毒(AAV)末端重复序列B和C回文近端的A茎的25bp序列中。通过证明在末端重复序列中没有其他序列的情况下，Rep68可以特异性地结合到含有25bp区域的合成寡核苷酸，从而最终证明了这一点。Rep78也能够与A茎识别元件结合，这从包含25bp序列的DNA亲和柱可以用于纯化Rep78的事实中得到证明。识别A茎中线性DNA序列的能力提供了一种机制，通过该机制可以将Rep蛋白定位在末端重复序列上，从而在末端分辨过程中只有正确的链在末端分辨位置(trs位点)。此外，计算机分析表明，在AAV的三个启动子区域中存在与A茎元件类似的序列。凝胶迁移率改变实验清楚地证明了P5启动子含有Rep结合序列。DNA酶保护实验表明，P5启动子内的Rep结合序列位于YY1启动子序列和TATA结合位点之间。这一位置立即暗示了一种机制，即Rep蛋白可以通过与YY1或TBP相互作用而作为P5转录的抑制或反式激活因子。此外，凝胶漂移实验表明，p19启动子也含有Rep结合位点。这两个启动子上游都存在Rep结合位点，提示这些结合位点可能参与AAV转录的协调调控。此外，我们还在pBR322 DNA中鉴定了一个异源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。在随附的报告中(D.M.McCarty，J.H.Ryan，S.Zlutukhin，X.周，和N.Muzyczka，J.Virol.68：4998-5006,1994)，我们检查了Rep与A茎位点和完整末端重复序列的相对亲和力。最后，我们还重新检查了Rep68和Rep78在不包含B和C回文或任何明显二级结构的底物中切割trs位点的能力。虽然需要更多的酶，但在含有Rep68和Rep78的这种底物上可以检测到trs内切酶活性。这一发现表明，A茎识别元件和功能的trs位点足以在trs位点进行位点特异性切割，并提供了一种机制，在AAV DNA复制过程中，预期不会有发夹的二聚体分子可能被加工成单体长度。这也意味着AAV末端重复是DNA复制的三方起源。为了有效地发挥功能，末端重复序列的三个元件是必需的：trs位点、A茎结合元件以及B和C回文。
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 cub 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. Although higher amounts of enzyme are necessary, trs endonuclease activity can be detected on such substrates with both Rep68 and Rep78. This finding suggests that the A stem recognition element and a functional trs site are sufficient for site specific cutting at the trs site and provides a mechanism by which dimer molecules, which are not expected to have hairpins, might be processed to monomer length during AAV DNA replication. It also means that the AAV terminal repeat is a tripartite origin for DNA replication. For efficient function, three elements of the terminal repeat are necessary: the trs site, the A stem-binding element, and the B and C palindromes.