Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease
Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease
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DOI：
10.1021/bi060423z
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发表时间：
2006-09-05
期刊：
影响因子：
2.9
通讯作者：
Wang, Yinsheng
中科院分区：
文献类型：
--
作者：
Gu, Chunang;Zhang, Qibin;Wang, Yinsheng
核苷酸切除修复 (NER) 是一种修复途径，可去除原核和真核细胞中的各种大体积 DNA 损伤。由氧化链内损伤引起的 DNA 螺旋结构的扰动可以使它们成为 NER 途径的良好底物。在这里，我们使用大肠杆菌NER酶，即UvrA、UvrB和UvrC，来检查携带三种不同氧化链内交联损伤（即G[8-5]C、G[8-5m]mC和G[8-5m]T）和两种二胸腺嘧啶光产物（即顺式， 顺式环丁烷嘧啶二聚体 (T[c, s]T) 和嘧啶(6-4) 嘧啶酮产物 (T[6-4]T)。我们的结果表明，T[6-4]T 是 UvrA 结合的最佳底物，其次是 G[8-5]C、G[8-5m]mC 和 G[8-5m]T，最后是 T[c, s]T。这些病灶的 UvrABC 切口效率与其 UvrA 结合亲和力一致：与 UvrA 的结合越强，切口率越高。此外，侧翼 DNA 序列似乎对 UvrA 与 G[8-5]C 的结合亲和力几乎没有影响，因为 AG[8-5]CA 仅略微优于 CG[8-5]CG。一致的是，这两个序列在切割率方面几乎没有差异。此外，我们研究了含有 G[8-5m]mC 或 G[8-5m]T 的十二聚体双链体的热稳定性，结果表明，这两个损伤使双链体不稳定，因为 37℃ 下双链体形成的自由能分别增加了约 5.4 和 3.6 kcal/mol。这些损伤引起的 DNA 螺旋不稳定在很大程度上与 UvrA 的结合亲和力和 UvrABC 的切割率相关。总而言之，这项研究的结果表明，氧化链内损伤可能是体内 NER 酶的底物。
Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis, syn-cyclobutane pyrimidine dimer (T[c, s]T) and the pyrimidine(6-4) pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c, s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.