Bertrand-Burggraf E, Selby C P, Hearst J E, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
J Mol Biol. 1991 May 5;219(1):27-36. doi: 10.1016/0022-2836(91)90854-y.
(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.
（A）BC核酸外切酶是由大肠杆菌的UvrA、UvrB和UvrC蛋白共同作用产生的酶活性。该酶能以不同效率从DNA中去除多种结构不同的加合物。为了解底物识别机制和酶特异性的基础，我们研究了这三个亚基与两种合成底物的相互作用，一种底物含有补骨脂素 - 胸腺嘧啶单加合物，另一种含有胸腺嘧啶二聚体。使用DNase I作为探针，我们发现UvrA在补骨脂素 - 胸腺嘧啶加合物周围形成一个33个碱基对的足迹，并且UvrA - UvrB形成一个45个碱基对的不对称足迹，其特征是在加合物5'端11个核苷酸处有一个超敏位点，且主要在损伤的3'侧受到保护。有利于UvrA从UvrA - UvrB - DNA复合物中解离的条件，如向反应混合物中加入过量未损伤的DNA，会导致形成一个19个碱基对的UvrB足迹。相比之下，在相似序列背景下的胸腺嘧啶二聚体未能引发UvrA、UvrA - UvrB或UvrB足迹，而是产生了一个相对较弱的、典型的UvrA - UvrB复合物的DNase I超敏位点。当加入UvrC时，UvrA从UvrA - UvrB - DNA复合物中解离会刺激两种底物的切割速率，这使我们得出结论：UvrA不是切割复合物的一部分，实际上它会干扰切割。加入UvrC后两种底物的切割程度（补骨脂素加合物为70%，胸腺嘧啶二聚体为20%）与UvrA - UvrB - DNA（即UvrB - DNA）复合物的形成程度成正比，表明底物识别发生在切割前的步骤。
