Needham-VanDevanter D R, Hurley L H, Reynolds V L, Theriault N Y, Krueger W C, Wierenga W
Nucleic Acids Res. 1984 Aug 10;12(15):6159-68. doi: 10.1093/nar/12.15.6159.
CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5'-CGGAGTTAGGGGCG-3') which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5'-TTA-3' was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 complexes formed with calf thymus DNA. The above 14mer was 5' end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5'-TTA-3' sequences.
CC - 1065是由泽链霉菌产生的一种强效抗肿瘤抗生素。该药物通过腺嘌呤的N - 3共价结合，并位于DNA的小沟内。先前的研究表明，CC - 1065与DNA中的腺嘌呤反应生成一种热不稳定产物，可用于揭示其序列特异性。这些研究还深入了解了一个DNA序列（5'-CGGAGTTAGGGGCG-3'），该序列应以明确的方式结合一分子的CC - 1065。这个序列在5'-TTA-3'序列中包含CC - 1065腺嘌呤结合位点，它与互补链一起被化学合成。CC - 1065与寡双链体反应生成一种加合物，该加合物保持B - DNA形式，其最终的圆二色光谱与用小牛胸腺DNA形成的CC - 1065复合物相似。上述14聚体用32P进行5'端标记，与互补链退火，与CC - 1065反应并加热。在测序凝胶上评估药物介导的链断裂。标记链中出现了一个单一的断裂，产生了一个迁移为8.5聚体的片段；随后用哌啶处理产生了一个迁移为7聚体的片段，这是根据CC - 1065在5'-TTA-3'序列中腺嘌呤处的已知结合预期的大小。
