Latham K A, Rajendran S, Carmical J R, Lee J C, Lloyd R S
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1071, USA.
Biochim Biophys Acta. 1996 Feb 8;1292(2):324-34. doi: 10.1016/0167-4838(95)00224-3.
Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.
核酸内切酶V是一种来自T4噬菌体的N-糖基化酶/裂解酶，可启动DNA中环丁烷嘧啶二聚体的修复。据报道，该酶在溶液中形成单体-二聚体平衡[Nickell和Lloyd（1991年），《生物化学》30, 8638]，尽管该酶仅在没有底物的情况下以单体形式结晶[Morikawa等人（1992年），《科学》256, 523]。在本研究中，使用分析凝胶过滤和沉降平衡技术严格表征了该酶在溶液中的缔合状态。与之前的报道相反，在100 mM KCl条件下，通过这两种技术发现核酸内切酶V在溶液中主要以单体形式存在；未观察到二聚化的证据。为了表征该酶在DNA靶位点的寡聚状态，将该酶与含有单个位点特异性嘧啶二聚体或四氢呋喃残基的寡核苷酸结合。通过在不同丙烯酰胺浓度下的非变性凝胶电泳分析这些复合物，以确定酶-DNA复合物的分子量。这些实验结果表明，核酸内切酶V以单体形式与含有环丁烷嘧啶二聚体和四氢呋喃位点的DNA结合。
