Dynamical Structural Change of Reconstituted Chromatin and Its Genetic Activity

近年来，单分子观察方法已被用来研究体外生物反应的分子机制。利用荧光显微镜对溶液中的单个DNA大分子进行了直接观察，发现DNA大分子的构象随环境条件的变化而变化。本研究的主要结果如下：(1)研究了肿瘤化疗药物柔红霉素对折叠的致密DNA被包裹在细胞大小的磷脂脂质体中的模型细胞系统的影响。在接触柔红霉素后，脂质体内致密的DNA逐渐拉长，然后被切割成一对片段。柔红霉素可以穿透双层脂膜，具有松解DNA堆积和切割双链DNA的潜在作用。我们的结果表明，细胞大小的脂质体作为活细胞的简单模型是有用的。(2)通过单分子荧光显微镜观察抗坏血酸对DNA高阶结构的影响，发现抗坏血酸在巨大的DNA分子中产生珍珠状结构，其中细长的部分和致密的部分沿着分子链共存。原子力显微镜的观察结果表明，致密部分呈松散堆积的构象。据报道，人类循环中的免疫细胞，如中性粒细胞、单核细胞和淋巴细胞，积累了毫摩尔浓度的抗坏血酸。因此，预计引起DNA大构象变化的抗坏血酸浓度可能具有生理意义。(3)我从高度致密的多核小体结构出发，研究了抗坏血酸对防止重组染色质DNA双链断裂的作用。

Recently, the methods of single-molecule observation have been utilized to study molecular mechanisms of biological reactions in vitro. With fluorescence microscopy, I have made direct observations of single giant DNA molecules in solution and have found that giant DNA molecules change their conformation depending on environmental conditions.The main results in the present study are summarized as follows.(1) I studied the effect of daunomycin, a cancer chemotherapeutic agent, on a model cellular system in which folded compact DNA was encapsulated inside a cell-sized phospholipid liposome. Following the exposure to daunomycin, the compact DNA inside a liposome was gradually elongated and then cut into a pair of fragments. It has become clear that daunomycin can penetrate the bilayer lipid membrane and has the potential effect to loosen the packing of DNA together with the activity to cut the double stranded DNA. Our results imply that cell-sized liposomes are useful as a simple model of living cells.(2) I examined the effect of ascorbic acid on the higher order structure of DNA through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant DNA molecules, where elongated and compact parts coexist along a molecular chain. The results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. It has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. Therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on DNA may be of physiological significance.(3) I investigated the effect of ascorbic acid on preventing DNA double-strand breaks in reconstituted chromatin in relation to the highly compacted polynucleosomal structure.