Orlowski R, Miller G
Department Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510-8064.
J Virol. 1991 Feb;65(2):677-86. doi: 10.1128/JVI.65.2.677-686.1991.
The Epstein-Barr virus (EBV) latent origin of plasmid replication (oriP) contains two essential regions, a family of repeats with 20 imperfect copies of a 30-bp sequence and a dyad symmetry element with four similar 30-bp repeats. Each of the repeats has an internal palindromic sequence and can bind EBNA 1, a protein that together with oriP constitutes the only viral element necessary for EBV maintenance and replication. Using single-strand-specific nucleases, we have probed plasmids containing oriP-derived sequences for the presence of secondary structural elements. Multiple single-stranded structures were detected within the oriP region. Of the two essential elements of oriP, the family of repeats seemed to extrude these structures at a much higher frequency than did sequences within the dyad symmetry region. Though negative supercoiling was found to stabilize the single-stranded structures, they showed significant stability even after linearization of the oriP plasmids. Two major single-stranded structures detected involved approximately 12 bp of DNA. These loci could be transiently unwound regions that form because of negative supercoiling and the high A + T content of this region of DNA, or they could be cruciform structures extruded within the palindromic sequences of oriP that may be important sites for protein-DNA interactions in the EBV oriP.
爱泼斯坦-巴尔病毒（EBV）质粒复制的潜伏性起始位点（oriP）包含两个必需区域，一个是由30个碱基对序列的20个不完全拷贝组成的重复序列家族，另一个是具有四个相似的30个碱基对重复序列的二元对称元件。每个重复序列都有一个内部回文序列，并且可以结合EBNA 1，EBNA 1是一种与oriP共同构成EBV维持和复制所需的唯一病毒元件的蛋白质。我们使用单链特异性核酸酶，探测了含有oriP衍生序列的质粒中二级结构元件的存在情况。在oriP区域内检测到多个单链结构。在oriP的两个必需元件中，重复序列家族似乎比二元对称区域内的序列以更高的频率挤出这些结构。虽然发现负超螺旋可稳定单链结构，但即使在oriP质粒线性化后，它们仍表现出显著的稳定性。检测到的两个主要单链结构涉及约12个碱基对的DNA。这些位点可能是由于负超螺旋和该DNA区域的高A+T含量而形成的瞬时解旋区域，或者它们可能是在oriP的回文序列内挤出的十字形结构，这可能是EBV oriP中蛋白质-DNA相互作用的重要位点。
