CHEMICAL PROBING OF B-ZJUNCTION IN DNA

DNA 中 B-Z 连接的化学探测

• 批准号: 63571042
• 负责人: NEGISHI Kazuo
• 金额: $ 1.34万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571042/
• 关键词: Bisulfite; Methoxyamine; Left-handed DNA; B-Zjunction; Monoclonal Antibody; High-salt; Chemical Modification; DNA polymerase; B型-Z型接合部; Z型DNA; モノクローナル抗体; 過マンガン酸カリウム; 亜硫酸塩; 高次構造; 5-メチルシトシン; 閉環状プラスミド

Bisulfite-methoxyamine mixture attacks (deoxy) cytidines in single-stranded region of nucleic acids. We have investigated the ability of this reagent as a chemical probe for B-Z junction of DNA. First, we studied the properties of the product N^4-methoxy-5, 6-dihydro(deoxy)cytidine 6-sulfonate. This product has two diastereomers. We have found that a commercially available monoclonal antibody against A4-methoxy-5,6- dihydro(deoxy) cytidine 6-sulfonate can bind specifically to the one disstereomer. Three double stranded oligonucleotides with blunt-end were synthesized. Two oligonucleotides contained cytosines at internal positions and 3'-end or 5'-end, respectively. The third oligonucleotide had cytosines only in internal positions. These oligonucleotides were modified with bisulfite-methoxyainine and analyzed with gel electrophoresis. The results showed that both 3' and 5' cytosines were accessible by the reagent. The gel was blotted onto a nylon membrane. The analysis of the blot with the monoclonal antibody revealed that 3'cytosine was modified to one diasteromer and 5' cytosine was modified to the odier diasteromer.Next, DNA containing Z-DNA was probed with bisulfite-methoxyamine. Alternating GC structure in pRW756 was made to Z form by supertwisting. This circular DNA was treated with bisulfite-methoxyamine. S1 nuclease digestion was employed to locate modification site. The results showed that cytosines of both junctions between oligo(GC) and B form DNA were modified. The modified cytosines were mapped in the sequence level by use of the pause of DNA polymerase action in opposite to the modified cytosines in the template. The modification of salt-induced B-Z junctions with bisulfite-methoxyamine gave similar results. These results indicates that B-Zjunction induced by supertwist and high salt have the similar structures.

亚硫酸氢盐-甲氧基胺混合物攻击核酸单链区域中的（脱氧）胞苷。我们研究了该试剂作为 DNA B-Z 连接化学探针的能力。首先，我们研究了产品N^4-甲氧基-5,6-二氢（脱氧）胞苷6-磺酸盐的性质。该产品有两种非对映体。我们发现市售的针对A4-甲氧基-5,6-二氢（脱氧）胞苷6-磺酸盐的单克隆抗体可以特异性结合一种非对映异构体。合成了三个具有平末端的双链寡核苷酸。两个寡核苷酸分别在内部位置和3'端或5'端含有胞嘧啶。第三个寡核苷酸仅在内部位置具有胞嘧啶。这些寡核苷酸用亚硫酸氢盐-甲氧基爱宁进行修饰，并用凝胶电泳进行分析。结果表明该试剂可接触到 3' 和 5' 胞嘧啶。将凝胶吸干到尼龙膜上。用单克隆抗体进行的印迹分析显示，3'胞嘧啶被修饰为一种非对映异构体，5'胞嘧啶被修饰为另一种非对映异构体。接下来，用亚硫酸氢盐-甲氧胺探测含有Z-DNA的DNA。 pRW756 中的交替 GC 结构通过超扭曲形成 Z 形。该环状DNA用亚硫酸氢盐-甲氧基胺处理。采用S1核酸酶消化来定位修饰位点。结果表明oligo(GC)和B型DNA之间的两个连接处的胞嘧啶均被修饰。通过使用与模板中修饰的胞嘧啶相反的DNA聚合酶作用的暂停，在序列水平上绘制修饰的胞嘧啶。用亚硫酸氢盐-甲氧基胺修饰盐诱导的 B-Z 连接也得到了类似的结果。这些结果表明超扭曲和高盐诱导的B-Z结具有相似的结构。

项目成果

K.Negishi: "Mutagenic nucleoside analog NA^4-aminocytidine:metabolism,incorporation into DNA,and mutagenesis in Escherichia coli" J.Bacteriol. 170. 5257-5262 (1988)

K.Negishi：“诱变核​​苷类似物 NA^4-氨基胞苷：大肠杆菌中的代谢、掺入 DNA 和诱变”J.Bacteriol。

K.Negishi: "Mutagenic nucleoside analog N^4-aminocytidine:metabolism,incorporation into DNA,and mutagenesis in Escherichia coli" J.Bacteriol.170. 5257-5262 (1988)

K.Negishi：“诱变核​​苷类似物 N^4-氨基胞苷：大肠杆菌中的代谢、掺入 DNA 和诱变”J.Bacteriol.170。

根岸和雄: "N^4-アミノシチジンによる突然変異の機構" 薬学雑誌. (1900)

Kazuo Negishi：“N^4-氨基胞苷引起的突变机制”制药杂志（1900）。

T.Bessho: "Spectrum of N^4-aminocytidine mutagenesis" J.Mol.Biol.205. 659-664 (1989)

T.Bessho：“N^4-氨基胞苷诱变光谱”J.Mol.Biol.205。

K. Negishi, K. Tamanoi, M. Ishii, M. Kawakami, Y. Yamashita and H. Hayatsu: "Mutagenic nucleoside analog N^4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli" J. Bacteriol.170. 5227-5262 (1989)

K. Negishi、K. Tamanoi、M. Ishii、M. Kawakami、Y. Yamashita 和 H. Hayatsu：“诱变核​​苷类似物 N^4-氨基胞苷：大肠杆菌中的代谢、掺入 DNA 和诱变”J. Bacteriol。