Lin J J, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
J Biol Chem. 1992 Sep 5;267(25):17688-92.
(A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.
大肠杆菌的（A）BC核酸外切酶通过水解修饰碱基5'端的第8个磷酸二酯键和3'端的第15个磷酸二酯键，从DNA上去除受损核苷酸。该活性源于UvrA、UvrB和UvrC蛋白的有序作用。UvrA的作用是帮助组装UvrB-DNA复合物，它不参与由UvrB和UvrC进行的实际切口反应。为了研究UvrC在核酸酶活性中的作用，在体外对该蛋白C端一半中的一组组氨酸、天冬氨酸和谷氨酸残基进行了诱变。研究了这些突变对体内紫外线抗性和体外切口活性的影响。H538F、D399A、D438A和D466A突变导致极度紫外线敏感性。用这些突变蛋白重构的酶进行正常的3'切口，但在5'切口活性方面完全有缺陷。我们的数据表明，UvrC通过一种机制进行5'切口，即三个羧酸盐与H538和一个Mg2+离子协同作用，促进活性位点水分子的亲核攻击。
