McCarty D M, Ryan J H, Zolotukhin S, Zhou X, Muzyczka N
Department of Microbiology, School of Medicine, University at Stony Brook, New York 11794.
J Virol. 1994 Aug;68(8):4998-5006. doi: 10.1128/JVI.68.8.4998-5006.1994.
We have characterized a Rep binding sequence which is within the A stem region of the adeno-associated virus terminal repeat (TR) and compared its affinity with that of the complete hairpinned TR for pure Rep68. Both the A stem and the complete TR substrates produced a complex pattern of protein-DNA complexes in which at least six different bound species could be distinguished. Competition experiments suggested that the dissociation constant for the A stem sequence is approximately 125-fold higher than that for the complete TR. The competition experiments also suggested that the average number of Rep molecules per TR substrate molecule under conditions of saturating substrate is 3.7:1, while for the A stem substrate, the ratio is 10:1. In spite of the apparent difference in protein-to-DNA ratio in the complexes, no major difference was seen in the mobility or the pattern of the protein-DNA complexes with the two kinds of substrates, suggesting that the difference in protein-to-DNA ratio was due to the lower stability of the A stem complex rather than the actual number of Rep molecules per DNA molecule. At least some of the difference in stability of the two kinds of complexes was due to the fact that the dissociation rate of the A stem substrate from the protein-DNA complexes was approximately fourfold faster than that of the complete TR. The dissociation rate curves for both substrates, however, were complex, suggesting that substrate was being released from at least two different kinds of protein-DNA complexes at different rates. In addition, we have analyzed binding to several substitution mutants within the A stem of the TR. A five-base mutant near the terminal resolution site (trs site) had little effect on binding. Two other mutants produced seven- or five-base substitutions within the 25-bp sequence of the A stem that had been identified in the accompanying report (D. M. McCarty, D. J. Pereira, I. Zolotukhin, X. Zhou, J. H. Ryan, and N. Muzyczka, J. Virol. 68:4988-4997, 1994) as essential for binding. Each of these mutants eliminated some but not all of the repeating GAGC motifs in the 25-bp A stem region. Both of these mutants completely abolished binding to the A stem substrate but only partially reduced binding in the context of the complete hairpinned TR. Furthermore, neither mutant altered the pattern of Rep-DNA complexes produced.(ABSTRACT TRUNCATED AT 400 WORDS)
我们鉴定了一个位于腺相关病毒末端重复序列（TR）A茎区域内的Rep结合序列，并比较了其与完整发夹状TR对纯Rep68的亲和力。A茎和完整的TR底物均产生了复杂的蛋白质-DNA复合物模式，其中至少可区分出六种不同的结合物种。竞争实验表明，A茎序列的解离常数比完整TR的解离常数高约125倍。竞争实验还表明，在底物饱和的条件下，每个TR底物分子的Rep分子平均数量为3.7:1，而对于A茎底物，该比例为10:1。尽管复合物中蛋白质与DNA的比例存在明显差异，但两种底物的蛋白质-DNA复合物在迁移率或模式上未观察到重大差异，这表明蛋白质与DNA比例的差异是由于A茎复合物的稳定性较低，而非每个DNA分子上实际的Rep分子数量。两种复合物稳定性的差异至少部分是由于A茎底物从蛋白质-DNA复合物中的解离速率比完整TR快约四倍。然而，两种底物的解离速率曲线都很复杂，表明底物正以不同速率从至少两种不同类型的蛋白质-DNA复合物中释放。此外，我们分析了TR的A茎内几个替代突变体的结合情况。靠近末端切割位点（trs位点）的一个五碱基突变体对结合影响很小。另外两个突变体在A茎的25 bp序列内产生了七碱基或五碱基替代，该序列在随附报告（D.M. McCarty、D.J. Pereira、I. Zolotukhin、X. Zhou、J.H. Ryan和N. Muzyczka，《病毒学杂志》68:4988 - 4997，1994）中已被确定为结合所必需。这些突变体中的每一个都消除了25 bp A茎区域中部分但并非全部的重复GAGC基序。这两个突变体都完全消除了与A茎底物的结合，但仅部分降低了在完整发夹状TR背景下的结合。此外，这两个突变体都未改变产生的Rep-DNA复合物模式。（摘要截断于400字）
