Sun D, Hurley L H
Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin 78712.
Anticancer Drug Des. 1992 Feb;7(1):15-36.
Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180 degrees out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC" (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC" can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.
非变性凝胶电泳分析表明，（+）-CC-1065的硬化和缠绕效应会对T4 DNA连接酶催化的共价修饰DNA的连接产生异常的近端和远端抑制作用。（+）-CC-1065是由泽链霉菌产生的一种强效抗肿瘤抗生素。这种药物通过与DNA中腺嘌呤的N3选择性结合，位于DNA的小沟中，并以高度序列选择性的方式发生反应。先前的研究（Lee等人，1991年）表明，（+）-CC-1065会使DNA产生弯曲和缠绕。DNA的弯曲和序列特异性是由（+）-CC-1065的烷基化“A”亚基介导的，而（+）-CC-1065的非烷基化B和C亚基与DNA小沟底部之间紧密的范德华接触则导致了DNA的缠绕。用（+）-CC-1065和结构相关药物对寡聚物进行共价修饰，会导致T4 DNA连接酶对共价加合物位点5'侧的非共价修饰链产生优先抑制作用，但共价修饰链的连接则增强。我们推测，对近端链连接的差异效应是由于药物诱导的缠绕和螺旋稳定效应，这种效应主要发生在加合物的5'侧。除了对连接的近端抑制作用外，我们还描述了T4 DNA连接酶活性的远端抑制作用，这种作用仅在药物修饰的寡聚物中出现，如果成功，在连接后会导致DNA产生180度异相弯曲。在这种情况下，用（+）-CC-1065修饰的两个25聚体或一个21聚体加一个29聚体的连接会受到抑制。这种远端连接是（+）-CC-1065及其导致DNA螺旋硬化的类似物所特有的。使用环化试验证明了（+）-CC-1065诱导的硬化效应，并且发现这与（+）-CC-1065的内边缘与小沟底部之间紧密的范德华接触有关，也与（+）-CC-1065类似物（+）-ABC"（阿多来新）的苯并呋喃部分有关。我们从这些研究中得出结论，这些药物分子的DNA缠绕和螺旋稳定效应可以显著影响T4 DNA连接酶介导的近端连接效率，并且（+）-CC-1065、（+）-AB'C'和（+）-ABC"异常的螺旋硬化效应可以稳定由药物修饰形成的弯曲DNA结构，从而导致远端连接酶抑制。
