Ward G K, Shihab-el-Deen A, Zannis-Hadjopoulos M, Price G B
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Exp Cell Res. 1991 Jul;195(1):92-8. doi: 10.1016/0014-4827(91)90503-m.
Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies with structural specificity for DNA cruciforms have been produced (Frappier et al. J. Mol. Biol. 193, 751, 1987). The effect of these antibodies, when introduced into permeabilized cells, was to increase overall DNA synthesis and relative copy number of genes (Zannis-Hadjopoulos et al. EMBO J. 7, 1837, 1988); this was interpreted to be a consequence of antibody stabilization of the cruciforms located at or near replication origins resulting in multiple initiations of DNA replication at a single site. Fluorescent labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary which then changes with progression through S-phase (Ward et al. Exp. Cell Res. 188, 235, 1990). In order to determine the relationship of cruciform distribution in DNA with the nuclear matrix/chromosomal scaffold, we assessed the susceptibility of DNA containing cruciforms to digestion with DNase I. The majority of the cruciforms detectable at G1/S and throughout the nucleus are readily digested by DNase, suggesting that cruciform structures may not be intimately associated with matrix proteins. The fraction that is resistant to DNase I appears associated with nuclear membrane and the nucleolus. No cruciforms could be detected in metaphase chromosomes; cruciforms either are not present or are inaccessible--buried in the scaffold. The absence of cruciforms from metaphase chromosomes would be consistent with the viewpoint that the cruciform in vivo is a transient structure dependent upon and interacting with proteins essential for replication or transcription.
十字形结构被认为是真核细胞中DNA复制起点处或其附近的潜在识别结构。已经制备了对DNA十字形结构具有结构特异性的单克隆抗体（弗拉皮尔等人，《分子生物学杂志》193卷，第751页，1987年）。当将这些抗体引入通透细胞时，其作用是增加总体DNA合成和基因的相对拷贝数（赞尼斯 - 哈佐普洛斯等人，《欧洲分子生物学组织杂志》7卷，第1837页，1988年）；这被解释为抗体稳定位于复制起点处或其附近的十字形结构，导致在单个位点多次启动DNA复制的结果。用抗十字形抗体对细胞核进行荧光标记，在停滞于G1/S边界的细胞中产生不均匀的荧光模式，然后随着S期的进展而变化（沃德等人，《细胞实验研究》188卷，第235页，1990年）。为了确定DNA中十字形结构的分布与核基质/染色体支架的关系，我们评估了含有十字形结构的DNA对DNase I消化的敏感性。在G1/S期及整个细胞核中可检测到的大多数十字形结构很容易被DNase消化，这表明十字形结构可能与基质蛋白没有紧密关联。对DNase I有抗性的部分似乎与核膜和核仁相关。在中期染色体中未检测到十字形结构；十字形结构要么不存在，要么无法接近——被埋在支架中。中期染色体中不存在十字形结构与体内十字形结构是一种依赖于复制或转录所必需的蛋白质并与之相互作用的瞬时结构这一观点相符。
