CROSS-LINKING OF DNA WITH TRIMETHYLPSORALEN IS A PROBE FOR CHROMATIN STRUCTURE
CROSS-LINKING OF DNA WITH TRIMETHYLPSORALEN IS A PROBE FOR CHROMATIN STRUCTURE
复制标题
DOI：
10.1016/0092-8674(77)90080-0
复制
发表时间：
1977-01-01
期刊：
影响因子：
64.5
通讯作者：
PARDUE, ML
中科院分区：
文献类型：
--
作者：
CECH, T;PARDUE, ML
Me3-补骨脂素（4,5'',8-三甲基补骨脂素）与 DNA 发生光化学反应，导致形成共价单加合物和链间交联。 DNA 在小鼠肝核内与 3H-me3-补骨脂素发生光反应，反应程度平均为 1 个共价结合的 me3-补骨脂素分子/114 或每 246 个碱基对 (bp)。光反应的细胞核用微球菌核酸酶消化，已知微球菌核酸酶会优先降解核小体（含有大约 200 bp DNA 的染色质亚基）之间的 DNA。无论细胞核是否与me3-补骨脂素反应，用微球菌核酸酶消化细胞核都会产生相同MW的DNA片段。当在细胞核中进行消化时，共价结合的 3H-me3-补骨脂素的酸溶解速率远大于本体 DNA，尽管在使用去蛋白 DNA 的对照实验中，含有 me3-补骨脂素的 DNA 区域消化得更慢。在消化极限，当 45% 的 DNA 降解为酸溶性时，92% 的 3H-me3-补骨脂素被微球菌核酸酶从染色质中释放出来。在整个核酸酶消化过程中，me3-补骨脂素与 DNA 片段共价结合，其分子量先前针对微球菌核酸酶消化产物进行了表征。细胞核中 me3-补骨脂素反应的主要位点可能是对微球菌核酸酶最敏感的 DNA，即核小体之间的 DNA。由于 me3-补骨脂素交联可以通过 EM 定位在 DNA 中，因此该探针提供了一种可能的方法，用于确定染色体蛋白沿高 MW DNA（例如转录单位）的体内位置。
Me3-psoralen (4,5'',8-trimethylpsoralen) undergoes a photochemical reaction with DNA, resulting in the formation of covalent monoadducts and interstrand cross-links. DNA was photoreacted with 3H-me3-psoralen inside mouse liver nuclei to the extent of 1 covalently bound me3-psoralen molecule/114 or per 246 base pairs (bp) on the average. The photoreacted nuclei were digested with micrococcal nuclease, which is known to degrade the DNA preferentially between nucleosomes (subunits of chromatin containing approximately 200 bp of DNA). The digestion of nuclei with micrococcal nuclease produced DNA fragments of the same MW whether or not the nuclei was reacted with me3-psoralen. The rate of acid solubilization of covalently bound 3H-me3-psoralen was much greater than that of the bulk DNA when the digestion was performed in nuclei, even though me3-psoralen-containing regions of DNA were digested more slowly in control experiments with deproteinized DNA. At the digestion limit, when 45% of the DNA was degraded to acid solubility, 92% of the 3H-me3-psoralen was released from chromatin by micrococcal nuclease. Throughout the nuclease digestion, the me3-psoralen was covalently bound to DNA fragments with the MW previously characterized for micrococcal nuclease digestion products. The major site for me3-psoralen reaction in nuclei is probably with the DNA most susceptible to micrococcal nuclease, that is, with the DNA between nucleosomes. Because me3-psoralen cross-links can be located in DNA by EM this probe provides a possible method for determining the in vivo location of chromosomal proteins along high MW DNA, such as a transcription unit.