Devchand P R, McGhee J D, van de Sande J H
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Nucleic Acids Res. 1993 Jul 25;21(15):3437-43. doi: 10.1093/nar/21.15.3437.
The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to investigate three different features of protein-DNA interactions. Complexes can be probed by simple protection experiments ('footprinting') or by two kinds of interference assays: a missing thymine site (MT-site) experiment and a missing thymine methyl site (MTM-site) experiment. The three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their respective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in certain cases, extend previous interpretations: for example, comparison of the results obtained with the two interference assays shows that formation of the lac repressor-operator complex requires interactions with the methyl group of one particular thymine residue (T-13) in the operator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the properties of UDG recommend it as a versatile and convenient method to investigate DNA-protein interactions both in vitro and possibly in vivo.
DNA修复酶尿嘧啶-DNA糖基化酶（UDG）可用于研究蛋白质-DNA相互作用的三个不同特征。复合物可以通过简单的保护实验（“足迹法”）或两种干扰测定法进行探测：胸腺嘧啶缺失位点（MT位点）实验和胸腺嘧啶甲基缺失位点（MTM位点）实验。使用已充分表征的λ阻遏物和乳糖阻遏物与其各自操纵子位点结合的体外系统来评估这三种探测方法。用UDG探测获得的结果与先前在相同系统上进行的探测实验结果非常吻合，并且在某些情况下扩展了先前的解释：例如，比较两种干扰测定法获得的结果表明，乳糖阻遏物-操纵子复合物的形成需要与操纵子中一个特定胸腺嘧啶残基（T-13）的甲基相互作用，但也需要与操纵子位置7、8、9、21、23和24处胸腺嘧啶碱基的其他部分相互作用。总体而言，UDG的特性使其成为一种通用且方便的方法，可用于体外甚至可能在体内研究DNA-蛋白质相互作用。
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