Strzelecka T E, Hayes J J, Clore G M, Gronenborn A M
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1995 Mar 7;34(9):2946-55. doi: 10.1021/bi00009a026.
Binding of purified phage Mu Ner protein to a series of DNA fragments was investigated in order to determine the length requirements for tight specific binding. Gel retardation experiments with wild-type 307 base pair (bp) Mu DNA and shorter, synthetic oligonucleotides were performed, and apparent dissociation constants (KappD) were determined from the half-saturation point. While Ner formed four complexes with the 307 bp DNA fragment, only one complex was observed with the shorter DNAs. The 50 and 30 bp fragments had KappD values of 5 and 20 nM, respectively. Ner binding was progressively weaker with decreasing size of the DNA fragments, with no binding observed for 12mers. The shortest DNA fragments which bound well were two 18 bp fragments for which KappD values were in the range of 50-100 nM. The stoichiometry of Ner complexes with the 30 and 18 bp fragments was determined using a modified Ferguson method. Ner was found to form a tetramer on the 30 bp DNA and a dimer on the 18 bp DNA, which makes the latter a good candidate for the study of a Ner-DNA complex by NMR. In order to clarify which DNA regions were important for Ner-DNA binding, hydroxyl radical footprinting was performed for a range of Ner concentrations from 30 to 500 nM. The footprint revealed that Ner contacts the DNA backbone every 12-13 bp, on both strands of the DNA. The order in which protected regions appeared with increasing protein concentration indicated that two Ner monomers bound to DNA simultaneously. A model of Ner binding to DNA is proposed on the basis of these results.
为了确定紧密特异性结合所需的长度要求，研究了纯化的噬菌体Mu Ner蛋白与一系列DNA片段的结合情况。使用野生型307碱基对（bp）的Mu DNA和更短的合成寡核苷酸进行凝胶阻滞实验，并从半饱和点确定表观解离常数（KappD）。虽然Ner与307 bp的DNA片段形成了四种复合物，但与较短的DNA仅观察到一种复合物。50 bp和三十bp的片段的KappD值分别为5 nM和20 nM。随着DNA片段大小的减小，Ner的结合逐渐减弱，12聚体未观察到结合。结合良好的最短DNA片段是两个18 bp片段，其KappD值在50 - 100 nM范围内。使用改良的弗格森方法确定了Ner与30 bp和18 bp片段的复合物的化学计量。发现Ner在30 bp的DNA上形成四聚体，在18 bp的DNA上形成二聚体，这使得后者成为通过核磁共振研究Ner - DNA复合物的良好候选物。为了阐明哪些DNA区域对Ner - DNA结合很重要，对30至500 nM范围内的一系列Ner浓度进行了羟基自由基足迹分析。足迹显示Ner在DNA的两条链上每隔12 - 13 bp与DNA主链接触。随着蛋白质浓度增加，受保护区域出现的顺序表明两个Ner单体同时与DNA结合。基于这些结果提出了Ner与DNA结合的模型。
