Forrow S M, Lee M, Souhami R L, Hartley J A
Department of Oncology, University College London Medical School, UK.
Chem Biol Interact. 1995 May 19;96(2):125-42. doi: 10.1016/0009-2797(94)03577-u.
The ability of the naturally occurring A/T specific DNA minor groove binders netropsin and diastamycin A and two synthetic G/C selective oligopeptide analogues (1 and 2), to interfere with the catalytic activity of restriction endonucleases has been investigated. Enzymes were chosen to have A/T rich (EcoRI, EcoRV) or G/C rich (BalI, NruI) recognition sequences. An agarose gel assay was used to measure the cleavage of 32P-labelled DNA and ligand-DNA binding data was obtained using methidium-propyl EDTA footprinting. Netropsin and distamycin bind at the recognition sites, and dose-dependently inhibited cleavage by, EcoRI and EcoRV, (EcoRI > EcoRV). They were also more effective at inhibiting the catalytic activity of BalI than either 1 or 2. NruI was inhibited by distamycin and 2, but not by netropsin or 1. DNA footprinting revealed that neither 1 or 2 bound to the BalI or NruI recognition sequences under the conditions used whereas netropsin and distamycin footprint at adjacent sites. 1 binds to two of the three recognition sequences for the enzyme Fnu4HI (GCNGC) in the fragment studied and was shown to inhibit DNA cleavage only at these two sites. 2 binds strongly to two GGGCTC sequences which are recognition sites for the enzyme BanII. In this case a pronounced stimulation of cleavage was observed in the presence of 2 over a wide dose range. The results indicate that enzyme inhibition does not necessarily result from simultaneous occupancy of a common site, or at nearby flanking sequences, and in some circumstances, a pronounced stimulation of enzyme cleavage can occur.
已经研究了天然存在的A/T特异性DNA小沟结合剂纺锤菌素和偏端霉素A以及两种合成的G/C选择性寡肽类似物（1和2）干扰限制性内切核酸酶催化活性的能力。选择具有富含A/T（EcoRI、EcoRV）或富含G/C（BalI、NruI）识别序列的酶。使用琼脂糖凝胶测定法测量32P标记的DNA的切割，并使用甲基丙基乙二胺四乙酸足迹法获得配体-DNA结合数据。纺锤菌素和偏端霉素在识别位点结合，并剂量依赖性地抑制EcoRI和EcoRV的切割（EcoRI>EcoRV）。它们在抑制BalI的催化活性方面也比1或2更有效。NruI被偏端霉素和2抑制，但不被纺锤菌素或1抑制。DNA足迹显示，在所使用的条件下，1或2都不与BalI或NruI识别序列结合，而纺锤菌素和偏端霉素在相邻位点形成足迹。1与所研究片段中酶Fnu4HI（GCNGC）的三个识别序列中的两个结合，并显示仅在这两个位点抑制DNA切割。2与两个GGGCTC序列强烈结合，这两个序列是酶BanII的识别位点。在这种情况下，在2存在的宽剂量范围内观察到切割的明显刺激。结果表明，酶抑制不一定是由于同时占据共同位点或附近侧翼序列导致的，并且在某些情况下，可能会出现酶切割的明显刺激。
