Carlson J O, Pfenninger O, Sinden R R, Lehman J M, Pettijohn D E
Nucleic Acids Res. 1982 Mar 25;10(6):2043-63. doi: 10.1093/nar/10.6.2043.
Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.
当光亲和药物三甲基补骨脂素在双链DNA的限制内切酶切割位点或其附近发生光结合时，该位点的切割会被阻断。一般来说，Hind III位点对失活的敏感性比所测试的其他限制位点高约15倍，尽管不同的Hind III位点的敏感性似乎会因位点附近的碱基序列而有所不同。Hind III位点可以通过两种方式失活；一种是完全阻断特定限制内切酶的作用，另一种是允许引入一个旋转点，使DNA超螺旋松弛而不产生双链断裂。核小体以及可能的其他蛋白质-DNA复合物可以保护其下的DNA序列免受三甲基补骨脂素的光结合，从而保护限制位点不被失活。这一特性可用于确定体内补骨脂素探针是否能接近特定位点，从而确定特定核苷酸序列是否与核小体相关。通过这个程序，我们获得了证据，即活的感染细胞中SV40 DNA上的核小体要么随机分布，要么分布在许多接近随机分布的离散交替位点上。
