Visse R, de Ruijter M, Moolenaar G F, van de Putte P
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
J Biol Chem. 1992 Apr 5;267(10):6736-42.
One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.
在UvrABC介导的切除修复过程中，最不为人所理解的步骤之一是对DNA损伤的识别。分离不同的反应中间体对于阐明其机制至关重要。本文描述了一种迁移率变动凝胶电泳分析方法，该方法可使这些中间体可视化。将含有特定顺铂加合物的DNA底物单独与UvrA孵育，或与UvrA和UvrB一起孵育后，观察到UvrA.DNA、UvrAB.DNA和UvrB.DNA复合物，这些复合物可用特异性抗体进行鉴定。在UvrB存在的情况下，当UvrA浓度较低时，仅观察到UvrB.DNA复合物。在相对较高的UvrA浓度下，发现了与UvrAB.DNA复合物相对应的条带以及其他非特异性条带。UvrAB.DNA复合物和UvrB.DNA复合物的DNase-I足迹非常相似，可保护约20个碱基。在UvrC存在的情况下，两种复合物的切割效率相当。UvrAB.DNA复合物和UvrB.DNA复合物均在特定顺铂加合物5'端的第8个磷酸二酯键处被切割。此外，UvrAB.DNA复合物也可在损伤位点5'端的第15个磷酸二酯键处被切割。结果表明，UvrB.DNA复合物是UvrC诱导切割的天然底物。
