Dynamical Structural Change of Reconstituted Chromatin and Its Genetic Activity

近年来，单分子观察方法已被用于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巨分子进行了直接观察，发现DNA巨分子的构象随环境条件的变化而变化。(1)我研究了道诺霉素（一种癌症化疗剂）对模型细胞系统的影响，在该系统中，折叠的紧凑DNA被封装在细胞大小的磷脂脂质体中。在暴露于柔红霉素后，脂质体内的紧密DNA逐渐伸长，然后切割成一对片段。已经清楚的是，柔红霉素可以穿透双层脂质膜，并且具有使DNA的包装松散的潜在作用以及切割双链DNA的活性。我们的研究结果表明，细胞大小的脂质体是有用的活细胞的一个简单的模型。(2)我通过荧光显微镜的单分子观察研究了抗坏血酸对DNA高级结构的影响，发现抗坏血酸在巨大的DNA分子中产生珍珠状结构，其中细长和紧凑的部分沿着分子链共存。原子力显微镜的观察结果表明，紧凑的部分采取松散堆积的构象。据报道，人循环免疫细胞，如嗜中性粒细胞、单核细胞和淋巴细胞，以毫摩尔浓度积累抗坏血酸。因此，预计诱导DNA发生较大构象变化的抗坏血酸浓度可能具有生理意义。(3)我研究了抗坏血酸对防止重组染色质中DNA双链断裂的影响，这与高度致密的多核小体结构有关。

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.