Myles G M, Hearst J E, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
Biochemistry. 1991 Apr 23;30(16):3824-34. doi: 10.1021/bi00230a004.
UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.
UvrA是DNA修复酶（A）BC核酸外切酶的ATP酶亚基。该蛋白质的氨基酸序列显示，除了两个锌指结构外，还有三对核苷酸结合基序，每对均由一个沃克A序列和一个沃克B序列组成。我们进行了位点特异性诱变、ATP酶动力学分析和核苷酸结合平衡测量，以将这些序列基序与活性相关联。在假定的A序列中用丙氨酸取代不变的赖氨酸表明，K37和K646而非K353参与ATP水解。相比之下，在B序列中位于D238、D513或D857位置的不变天冬氨酸被天冬酰胺取代，对该蛋白质的体内活性影响很小。核苷酸结合研究表明，ADP/UvrA单体的化学计量比为0.5，而对野生型和突变型蛋白质的动力学测量表明，UvrA的活性形式是具有2个催化位点的二聚体，在ADP存在下以正协同方式相互作用；K37而非K646的诱变减弱了这种协同性。K37A突变体中ATP酶活性丧失约75%，K646A突变体中为86%，K37A-K646A双突变体中为95%。这些氨基酸取代对UvrA与受损DNA的特异性结合仅有微小影响，但极大地降低了其将UvrB递送至损伤位点的能力。我们发现，这些突变型UvrA蛋白的UvrB加载活性不足是由于它们无法以（UvrA）2（UvrB）1复合物的形式与UvrB结合。我们得出结论，UvrA形成一个具有两个涉及K37和K646的ATP酶结构域的二聚体，并且ATP水解所执行的功能是将UvrB递送至DNA上的损伤位点。
