Huang J C, Svoboda D L, Reardon J T, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3664-8. doi: 10.1073/pnas.89.8.3664.
By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate consisting of a plasmid containing four thymidine dimers at unique locations, and deoxyribonucleoside 5'-[alpha-thio]triphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by I2, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer.
通过使用一种能够进行核苷酸切除修复的无细胞人源系统、一个由在独特位置含有四个胸腺嘧啶二聚体的质粒组成的合成底物，以及用于修复合成的脱氧核糖核苷5'-[α-硫代]三磷酸，我们获得了含有具有硫代磷酸酯键的修复补丁的DNA片段。基于这些键对核酸外切酶III消化的抗性及其对I2切割的敏感性，我们能够将修复补丁的边界精确到单核苷酸分辨率，并发现了一个具有清晰边界的不对称补丁。胸腺嘧啶二聚体以27至29个核苷酸的寡聚物形式释放，这一发现证实了修复补丁是由填补切除核酸酶产生的缺口而非切口平移产生的。
