Mocquet Vincent, Kropachev Konstantin, Kolbanovskiy Marina, Kolbanovskiy Alexander, Tapias Angels, Cai Yuqin, Broyde Suse, Geacintov Nicholas E, Egly Jean-Marc
Chemistry Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Strasbourg, France.
EMBO J. 2007 Jun 20;26(12):2923-32. doi: 10.1038/sj.emboj.7601730. Epub 2007 May 24.
Benzo[a]pyrene (B[a]P), a known environmental pollutant and tobacco smoke carcinogen, is metabolically activated to highly tumorigenic B[a]P diol epoxide derivatives that predominantly form N(2)-guanine adducts in cellular DNA. Although nucleotide excision repair (NER) is an important cellular defense mechanism, the molecular basis of recognition of these bulky lesions is poorly understood. In order to investigate the effects of DNA adduct structure on NER, three stereoisomeric and conformationally different B[a]P-N(2)-dG lesions were site specifically incorporated into identical 135-mer duplexes and their response to purified NER factors was investigated. Using a permanganate footprinting assay, the NER lesion recognition factor XPC/HR23B exhibits, in each case, remarkably different patterns of helix opening that is also markedly distinct in the case of an intra-strand crosslinked cisplatin adduct. The different extents of helix distortions, as well as differences in the overall binding of XPC/HR23B to double-stranded DNA containing either of the three stereoisomeric B[a]P-N(2)-dG lesions, are correlated with dual incisions catalyzed by a reconstituted incision system of six purified NER factors, and by the full NER apparatus in cell-free nuclear extracts.
苯并[a]芘（B[a]P）是一种已知的环境污染物和烟草烟雾致癌物，经代谢活化后生成具有高度致瘤性的B[a]P二醇环氧化物衍生物，这些衍生物主要在细胞DNA中形成N(2)-鸟嘌呤加合物。尽管核苷酸切除修复（NER）是一种重要的细胞防御机制，但对这些大分子损伤的识别分子基础仍知之甚少。为了研究DNA加合物结构对NER的影响，将三种立体异构且构象不同的B[a]P-N(2)-dG损伤位点特异性地掺入相同的135聚体双链体中，并研究它们对纯化的NER因子的反应。使用高锰酸盐足迹分析，NER损伤识别因子XPC/HR23B在每种情况下都表现出明显不同的解旋模式，这种模式在链内交联顺铂加合物的情况下也明显不同。三种立体异构的B[a]P-N(2)-dG损伤中的任何一种在双链DNA上，XPC/HR23B的螺旋扭曲程度不同以及整体结合情况的差异，与由六种纯化的NER因子组成的重组切割系统以及无细胞核提取物中的完整NER装置催化的双重切割相关。
