Steinmetzer K, Zannis-Hadjopoulos M, Price G B
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
J Mol Biol. 1995 Nov 17;254(1):29-37. doi: 10.1006/jmbi.1995.0596.
Cruciform DNA structure, as a structural feature, has been associated with regulation of transcription, recombination and replication. Previously used to successfully modify DNA replication and affinity-purify origins and autonomously replicating sequences. Using enzyme protection assays, their binding activity has been localized to the base (elbow) of the cruciform stem. We report here the hydroxyl radical footprinting of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plasmids, identical except for two centrally located palindromes of different sequence. The footprinting was performed at near-physiological salt concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the four-way junction (elbows) of the stable cruciform. The binding of the antibody seems also to cause associated structural distortions in the heteroduplex, which generally result in greater sensitivity to hydroxyl radicals at the tips of the cruciforms. The data are consistent to hydroxyl radicals at the tips of the cruciforms. The data are consistent with the binding of a single antibody to an antigen-combining site. The results of this study compare favourably with the hydroxyl radical footprinting studies reported recently for a human cruciform binding protein (CBP), which binds at the base of the stem-loop structure and causes similar distortions of the stable cruciform structure. These studies indicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of this structural determinant with DNA replication and the existence of a novel cellular protein, CBP, of similar binding specificity as the antibody specificity support a role for cruciforms as important regulatory recognition signals in replication.
十字形DNA结构作为一种结构特征，与转录、重组和复制的调控相关。此前已成功用于修饰DNA复制以及亲和纯化起源和自主复制序列。通过酶保护分析，发现它们的结合活性定位于十字形茎的基部（弯头）。我们在此报告2D3（κIgG1）抗十字形单克隆抗体对由两个质粒片段异源双链形成的稳定十字形结构的羟基自由基足迹分析，这两个质粒除了两个位于中心的不同序列回文序列外完全相同。足迹分析是在接近生理盐浓度的条件下进行的，这种条件有利于十字形的堆叠X结构。我们的数据表明抗体的结合发生在稳定十字形的四向连接点（弯头）。抗体的结合似乎也会在异源双链中引起相关的结构扭曲，这通常会导致十字形末端对羟基自由基更敏感。这些数据与十字形末端的羟基自由基一致。数据与单个抗体与抗原结合位点的结合一致。本研究结果与最近报道的关于人十字形结合蛋白（CBP）的羟基自由基足迹分析研究相比具有优势，该蛋白结合在茎环结构的基部并导致稳定十字形结构的类似扭曲。这些研究表明十字形的四向连接点具有某些独特的抗原性结构特性；这种结构决定因素与DNA复制的关联以及与抗体特异性具有相似结合特异性的新型细胞蛋白CBP的存在支持十字形作为复制中重要调控识别信号的作用。
