Zou Y, Walker R, Bassett H, Geacintov N E, Van Houten B
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Biol Chem. 1997 Feb 21;272(8):4820-7. doi: 10.1074/jbc.272.8.4820.
The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.
大肠杆菌的UvrB和UvrC蛋白在核苷酸切除修复过程中的DNA损伤处理和切口形成中发挥关键作用。为了研究这些过程中形成的DNA结构要求和蛋白质-DNA中间体，构建了苯并[a]芘二醇环氧化物损伤的和结构特异性的50碱基对底物。含有预先存在的3'切口的DNA片段在加合物的5'端被快速有效地切开。凝胶迁移率变动分析表明，该底物支持UvrA从UvrB-DNA复合物中解离，从而导致有效切口。用含有围绕苯并[a]芘二醇环氧化物加合物的内部非互补11碱基区域的DNA片段进行的实验表明，UvrABC核酸酶结合和切口形成不需要完全双链的DNA。在没有UvrA的情况下，UvrB（UvrC）与含有3'切口的11碱基非互补区域（Y底物）结合，形成稳定的蛋白质-DNA复合物（解离常数约为5-10 nM）。该复合物的形成绝对依赖于UvrC。向该复合物中添加ATP，但不添加腺苷5'-（β，γ-亚氨基三磷酸）或腺苷5'-（β，γ-亚甲基）三磷酸，会在双链-单链交界处的5'端切开三或四个核苷酸。天然UvrB的ATP酶活性在与UvrC相互作用时被激活，并通过添加Y底物进一步增强。即使没有DNA损伤，这种Y结构也会被切开。因此，UvrBC复合物是一种结构特异性的、ATP依赖的内切核酸酶。
