Suh D, Povirk L F
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
Biochemistry. 1997 Apr 8;36(14):4248-57. doi: 10.1021/bi962957d.
Although the binding of various forms of bleomycin to DNA has been studied extensively, the transient nature of the activated bleomycin species which ultimately attacks DNA has largely precluded direct examination of its physical interactions with DNA. In an attempt to map the minimum binding site required for this species to effect DNA cleavage, several oligonucleotide duplexes were synthesized, each of which contained a single N2-isobutyrylguanine moiety at a specific position in the sequence. These duplexes were end-labeled, and sequence-specific bleomycin-induced cleavage was assessed in each strand of each duplex. Isobutyrylguanine substitution immediately 5' to a primary bleomycin target site suppressed bleomycin-induced cleavage by more than 10-fold. Substitution two bases 5' to a target site suppressed cleavage by about 4-fold, and substitution directly opposite the target site suppressed cleavage by 7-fold. Substitution immediately 3' to the target site, or at other more distant positions 3' or 5', had little or no effect. In cases where cleavage at a primary site was strongly suppressed, cleavage at the corresponding secondary site (the putative site of the second break in a bleomycin-induced double-strand break) was also inhibited, even when the secondary site was several bases away from the isobutyrylguanine substitution. The results suggest that the binding site required for bleomycin-induced DNA cleavage spans a region of approximately 2 or 3 bp in the minor groove, including the base associated with the sugar attacked and one or two bases to its 5' side. Computer-based molecular modeling indicated that these results are consistent with the predictions of recently proposed models in which the bithiazole is intercalated immediately 3' to the cleavage site, and the iron coordination site binds in the minor groove immediately 5' to the cleavage site. Both the empirical data and the modeling studies suggest that N2-isobutyrylguanine substitution effectively blocks the minor groove, but without significantly disturbing DNA secondary structure. Thus, it is proposed that site-specific incorporation of N2-isobutyrylguanine may provide a general method for mapping binding sites of minor groove-binding ligands on DNA.
尽管对博来霉素各种形式与DNA的结合进行了广泛研究，但最终攻击DNA的活化博来霉素物种的瞬态性质在很大程度上排除了对其与DNA物理相互作用的直接研究。为了绘制该物种实现DNA切割所需的最小结合位点图谱，合成了几种寡核苷酸双链体，每个双链体在序列中的特定位置都含有一个单一的N2-异丁酰鸟嘌呤部分。这些双链体进行了末端标记，并评估了每个双链体每条链中序列特异性博来霉素诱导的切割。在主要博来霉素靶位点紧邻5'端的异丁酰鸟嘌呤取代使博来霉素诱导的切割抑制了10倍以上。在靶位点5'端两个碱基处的取代使切割抑制约4倍，而在靶位点正对面的取代使切割抑制7倍。在靶位点紧邻3'端或在3'或5'端其他更远位置的取代几乎没有影响。在主要位点的切割被强烈抑制的情况下，相应二级位点（博来霉素诱导的双链断裂中第二个断裂的假定位点）的切割也受到抑制，即使二级位点距离异丁酰鸟嘌呤取代有几个碱基。结果表明，博来霉素诱导DNA切割所需的结合位点在小沟中跨越约2或3个碱基对的区域，包括与被攻击糖相关的碱基及其5'侧的一个或两个碱基。基于计算机的分子建模表明，这些结果与最近提出的模型预测一致，在这些模型中，双噻唑在切割位点紧邻3'端插入，铁配位位点在切割位点紧邻5'端结合在小沟中。经验数据和建模研究均表明，N2-异丁酰鸟嘌呤取代有效地阻断了小沟，但没有显著干扰DNA二级结构。因此，有人提出N2-异丁酰鸟嘌呤的位点特异性掺入可能为绘制小沟结合配体在DNA上的结合位点提供一种通用方法。
