Zou Y, Liu T M, Geacintov N E, Van Houten B
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555, USA.
Biochemistry. 1995 Oct 17;34(41):13582-93. doi: 10.1021/bi00041a038.
Oligonucleotides containing site-specifically-modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. These substrates were used in reactions with the Escherichia coli UvrABC nuclease system. The interaction of the UvrA2 and UvrA2B complexes with these four stereoisomers was probed using DNase I footprinting and gel mobility shift assays. DNase I digestion of substrates containing each stereoisomer of BPDE displayed a unique pattern which was consistent with the known structure of these DNA adducts. UvrA and UvrA2B appeared to interact very similarly with all four substrates. Binding of UvrA2 to these substrates produced a 33-bp footprint, and the UvrB--DNA complex resulted in footprint of 24 bp. The UvrABC nuclease system produced bimodal incisions at the eighth phosphate 5' and the fifth, sixth, or seventh phosphate 3' to the modified guanine. The variation of the 3' incision site was linked to the stereochemistry and orientation of the BPDE adduct. For example, the 3' incision of the 50-bp duplex containing (-)-trans-BPDE-N2-guanine was inhibited at the fifth phosphate. UvrABC nuclease incision kinetics revealed a hierarchy of specificity. The intercalative cis isomers were incised more efficiently than the corresponding trans isomers which lie in the minor groove. The (+) enantiomers were incised more efficiently than the (-) form for both cis and trans isomers. These observations reveal that UvrABC nuclease recognition and incision are directly influenced by the conformation of the DNA adduct.
将含有位点特异性修饰的N2-鸟嘌呤（+）-反式、（-）-反式、（+）-顺式和（-）-顺式BPDE加合物的寡核苷酸连接到50个碱基对的DNA片段中。这些底物用于与大肠杆菌UvrABC核酸酶系统的反应。使用DNase I足迹法和凝胶迁移率变动分析来探究UvrA2和UvrA2B复合物与这四种立体异构体的相互作用。对含有BPDE每种立体异构体的底物进行DNase I消化显示出独特的模式，这与这些DNA加合物的已知结构一致。UvrA和UvrA2B似乎与所有四种底物的相互作用非常相似。UvrA2与这些底物的结合产生了一个33 bp的足迹，而UvrB-DNA复合物导致了一个24 bp的足迹。UvrABC核酸酶系统在修饰鸟嘌呤5'端的第八个磷酸和3'端的第五、第六或第七个磷酸处产生双峰切口。3'切口位点的变化与BPDE加合物的立体化学和方向有关。例如，含有（-）-反式-BPDE-N2-鸟嘌呤 的50 bp双链体的3'切口在第五个磷酸处受到抑制。UvrABC核酸酶的切口动力学揭示了特异性的层次结构。嵌入的顺式异构体比位于小沟中的相应反式异构体更有效地被切割。对于顺式和反式异构体，（+）对映体比（-）形式更有效地被切割。这些观察结果表明，UvrABC核酸酶的识别和切割直接受到DNA加合物构象的影响。
