Snowden A, Kow Y W, Van Houten B
Department of Pathology, University of Vermont, Burlington 05405.
Biochemistry. 1990 Aug 7;29(31):7251-9. doi: 10.1021/bi00483a013.
Using oligonucleotide synthesis, we demonstrate a rapid and efficient method for the construction of DNA duplexes containing defined DNA lesions at specific positions. These DNA lesions include apyrimidinic sites, reduced apyrimidinic sites, and base-damage analogues consisting of O-methyl- or O-benzylhydroxylamine-modified apyrimidinic sites. A 49 base pair DNA duplex containing these lesions was specifically incised by the UvrABC nuclease complex. The incision sites occurred predominantly at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion. Multiple incisions were observed 3' to the lesion. The extent of DNA incisions was base-damage analogues greater than reduced apyrimidinic sites greater than apyrimidinic sites. Introduction of 3' or 5' nicks at the site of a base-damage analogue by treatment of these substrates with either endonuclease III or endonuclease IV reduced, but did not abolish, subsequent incision by the UvrABC complex, whereas introduction of a 3' nick at an abasic site increased the incision efficiency of the UvrABC complex. These data demonstrate a convergence of base and nucleotide excision repair pathways in the removal of specific base damages.
通过寡核苷酸合成，我们展示了一种快速有效的方法，用于构建在特定位置含有特定DNA损伤的DNA双链体。这些DNA损伤包括无嘧啶位点、还原型无嘧啶位点以及由O-甲基或O-苄基羟胺修饰的无嘧啶位点组成的碱基损伤类似物。一种含有这些损伤的49个碱基对的DNA双链体被UvrABC核酸酶复合物特异性切割。切割位点主要出现在损伤位点5'端的第八个磷酸二酯键和3'端的第五个磷酸二酯键处。在损伤位点3'端观察到多个切割。DNA切割的程度为碱基损伤类似物大于还原型无嘧啶位点大于无嘧啶位点。用核酸内切酶III或核酸内切酶IV处理这些底物，在碱基损伤类似物位点引入3'或5'切口，可降低但不能消除随后UvrABC复合物的切割，而在无碱基位点引入3'切口则提高了UvrABC复合物的切割效率。这些数据表明在去除特定碱基损伤时碱基切除修复和核苷酸切除修复途径存在趋同现象。
