Han F X, Hurley L H
Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
Biochemistry. 1996 Jun 18;35(24):7993-8001. doi: 10.1021/bi960251d.
The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin. (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These unexpected results suggest that when T-antigen binds to the SV40 origin the AT tract is in a straight DNA conformation. High-resolution DNase I footprinting experiments indicate that at least three helically in-phase T-antigen binding sites exist in the GC box region located immediately downstream of the AT tract. The binding of T-antigen enhances the reactivity of (+)-CC-1065 to the two 5'-AGTTA(asterisk) (the asterisk indicates the covalent bonding site) drug modification sites in the GC box region, demonstrating that these sites are in a bent conformation. In contrast, T-antigen inhibited the reactivity of pluramycin at sequences within the GC box region that are known not to bind T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T-antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.
用三种与DNA反应的抗肿瘤药物：（+）-CC-1065、吡唑啉酮和腐草霉素，探究了T抗原诱导的SV40复制起点的结构变化。（+）-CC-1065是一种N3腺嘌呤小沟烷基化剂，它能选择性地与具有弯曲构象的富含AT的DNA序列发生反应；吡唑啉酮也与富含AT的序列的小沟发生反应，但对一种构象具有选择性；吡唑啉酮也与富含AT的序列的小沟发生反应，但对直链DNA构象具有选择性。腐草霉素是一种嵌入型鸟嘌呤烷基化剂，其反应活性会因DNA小沟和/或大沟的解旋而增加，因压缩而降低。我们发现，虽然T抗原的结合降低了（+）-CC-1065使SV40复制起点中的AT序列烷基化的能力，但它并不干扰同一序列的吡唑啉酮修饰。这些意外的结果表明，当T抗原与SV40起点结合时，AT序列处于直链DNA构象。高分辨率DNase I足迹实验表明，在紧接AT序列下游的GC框区域中至少存在三个螺旋同相的T抗原结合位点。T抗原的结合增强了（+）-CC-1065对GC框区域中两个5'-AGTTA（星号表示共价结合位点）药物修饰位点的反应活性，表明这些位点处于弯曲构象。相反，T抗原抑制了腐草霉素对GC框区域内已知不结合T抗原的序列的反应活性。这些数据与DNase I足迹结果相结合，表明T抗原结合诱导了DNA的构象变化，不再有利于腐草霉素的嵌入。基于我们的结果，我们提出T抗原紧密结合于SV40复制起点AT序列的上游区域，形成双六聚体。在下游区域，T抗原与GC框区域中螺旋同相位点的结合诱导DNA向与天然AT序列弯曲相反的方向弯曲，同时将天然弯曲的AT序列DNA转变为直链构象。
