二价铁配合物通过“水解”机制对pBR322进行双链切割。

Double-stranded cleavage of pBR322 by a diiron complex via a "hydrolytic" mechanism.

作者信息

Schnaith L M, Hanson R S, Que L

机构信息

Department of Chemistry, University of Minnesota, Minneapolis 55455.

出版信息

Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):569-73. doi: 10.1073/pnas.91.2.569.

Treatment of plasmid pBR322 with Fe2-(HPTB)(OH)(NO3)4(HPTB = N,N,N',N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopr opane) and H2O2 or O2 and a reductant (dithiothreitol or ascorbate) results in double-stranded cleavage of the plasmid. The linearization of supercoiled pBR322 by this complex is not inhibited by hydroxyl radical scavengers. On the other hand, the linearized pBR322 is efficiently religated by T4 DNA ligase, and the presence of 3'-OH and 5'-OPO3 ends is corroborated by 3'- and 5'-end-labeling studies. These observations indicate that cleavage results from hydrolysis of the DNA-phosphate backbone, which is proposed to occur by nucleophilic attack of the bound peroxide on the phosphodiester. Double-stranded cleavage by the Fe2(HPTB)(OH)(NO3)4/H2O2 adduct preferentially occurs between bp 3489 and 3485 of pBR322.

用Fe2-(HPTB)(OH)(NO3)4（HPTB = N,N,N',N'-四（2-苯并咪唑基甲基）-2-羟基-1,3-二氨基丙烷）与H2O2或O2以及一种还原剂（二硫苏糖醇或抗坏血酸盐）处理质粒pBR322会导致质粒的双链断裂。这种复合物对超螺旋pBR322的线性化作用不受羟基自由基清除剂的抑制。另一方面，线性化的pBR322能被T4 DNA连接酶有效地重新连接，并且3'-末端标记和5'-末端标记研究证实了3'-OH和5'-OPO3末端的存在。这些观察结果表明，断裂是由DNA-磷酸主链的水解引起的，推测这是通过结合的过氧化物对磷酸二酯的亲核攻击而发生的。Fe2(HPTB)(OH)(NO3)4/H2O2加合物引起的双链断裂优先发生在pBR322的3489和3485碱基对之间。