Waterloh K, Fox K R
Department of Physiology and Pharmacology, University of Southampton, United Kingdom.
J Biol Chem. 1991 Apr 5;266(10):6381-8.
The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.
利用多种对DNA结构敏感的探针，通过足迹分析研究了放线菌素对克隆到pUC19的SmaI位点中含有序列(AT)5GC(AT)5、(TA)5GC(TA)5、A9GCT9和T9GCA9的DNA片段结构的影响。在每种情况下，在中央GC位点周围都发现了清晰的足迹。含有交替AT的片段的DNase I切割显示在ApT处的切割比在TpA处大得多；在放线菌素存在下，虽然这种偏好得以保留，但在最接近的TpA步骤处切割效率大幅提高。通常非常低效的A和T同聚物区域中的DNase I切割因药物结合而大大增强。对于T9GCA9，增强作用向两个方向传播，而在A9GCT9中，变化仅在GC位点的5'侧发现。用微球菌核酸酶和DNase II进行的类似实验证实了这些结果。在GC附近的腺嘌呤处发现对焦碳酸二乙酯的敏感性略有增加。在4℃进行的实验表明，构象变化是药物结合的必然结果。
