Snowden A, Van Houten B
Department of Pathology, University of Vermont, Burlington 05405.
J Mol Biol. 1991 Jul 5;220(1):19-33. doi: 10.1016/0022-2836(91)90378-j.
The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.
大肠杆菌核苷酸切除修复的切口步骤由UvrABC核酸酶复合物介导。我们之前已经表明，UvrABC核酸酶切割无嘧啶（AP）位点的效率比邻苄基羟胺修饰的无嘧啶（BA）位点低。为了研究这些差异，进行了定量DNase I足迹滴定研究。尽管这两种损伤的切口程度不同，但AP位点（Kd = 6×10⁻⁹ M）和体积更大的BA损伤（Kd = 14×10⁻⁹ M）的UvrA结合等温线相似。还发现，切割前的UvrA₂B复合物与AP和BA底物的相对结合亲和力存在显著差异，估计的表观平衡解离常数（Kd）分别为4×10⁻⁹ M和80×10⁻⁹至120×10⁻⁹ M。这些结果表明，切口效率与UvrA结合亲和力无关，而是UvrA₂B复合物与DNA损伤位点之间相互作用的直接结果。还表明，高浓度的UvrA对UvrABC核酸酶反应具有抑制作用。
