Christ F, Steuer S, Thole H, Wende W, Pingoud A, Pingoud V
Institut für Biochemie Fachbereich 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
J Mol Biol. 2000 Jul 21;300(4):867-75. doi: 10.1006/jmbi.2000.3872.
We have synthesized different oligodeoxynucleotides carrying, in single positions of the >36 bp recognition site of PI-SceI, photoreactive base analogues (5-iododeoxypyrimidines) or phosphate modifications (p-azidophenacylphosphorothioates) and used them in photocross-linking experiments with PI-SceI to probe the protein-DNA interface of the specific complex between the homing endonuclease PI-SceI and its DNA substrate. One base-specific and several backbone-specific cross-links were analyzed in detail: the cross-linking positions were identified by Edman degradation of isolated cross-linked peptidexoligodeoxynucleotide adducts and confirmed by site-directed mutagenesis. Based on these results and the crystal structure of PI-SceI, a model for the structure of the PI-SceIxDNA complex is proposed.
我们合成了不同的寡脱氧核苷酸，这些寡脱氧核苷酸在PI-SceI的>36 bp识别位点的单个位置携带光反应性碱基类似物（5-碘脱氧嘧啶）或磷酸修饰（对叠氮苯甲酰硫代磷酸酯），并将它们用于与PI-SceI的光交联实验，以探测归巢内切酶PI-SceI与其DNA底物之间特定复合物的蛋白质-DNA界面。详细分析了一个碱基特异性和几个主链特异性交联：通过对分离的交联肽-寡脱氧核苷酸加合物进行埃德曼降解确定交联位置，并通过定点诱变进行确认。基于这些结果和PI-SceI的晶体结构，提出了PI-SceI-xDNA复合物结构的模型。
