Gunz D, Hess M T, Naegeli H
Institute of Pharmacology and Toxicology, University of Zürich-Tierspital, Winterthurerstrasse 260, 8057 Zürich, Switzerland.
J Biol Chem. 1996 Oct 11;271(41):25089-98. doi: 10.1074/jbc.271.41.25089.
The mechanism by which mammalian nucleotide excision repair (NER) detects a wide range of base lesions is poorly understood. Here, we tested the ability of human NER to recognize bulky modifications that either destabilize the DNA double helix (acetylaminofluorene (AAF) and benzo[a]pyrene diol-epoxide (BPDE) adducts, UV radiation products) or induce opposite effects by stabilizing the double helix (8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 adducts). We constructed plasmid DNA carrying a defined number of each of these adducts and determined their potential to sequester NER factors contained in a human cell-free extract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. This novel approach showed differences of more than 3 orders of magnitude in the efficiency by which helix-destabilizing and helix-stabilizing adducts sequester NER factors. For example, AAF modifications were able to compete with the NER substrate approximately 1740 times more effectively than 8-MOP adducts. The sequestration potency decreased with the following order of adducts, AAF > UV >/= BPDE > 8-MOP > anthramycin, CC-1065. A strong preference for helix-destabilizing lesions was confirmed by monitoring the formation of NER patches at site-specific adducts with either AAF or CC-1065. This comparison based on factor sequestration and repair synthesis indicates that human NER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition involves thermodynamic probing of the duplex.
哺乳动物核苷酸切除修复（NER）检测多种碱基损伤的机制目前仍知之甚少。在此，我们测试了人类NER识别大分子修饰的能力，这些修饰要么使DNA双螺旋不稳定（乙酰氨基芴（AAF）和苯并[a]芘二醇环氧化物（BPDE）加合物、紫外线辐射产物），要么通过稳定双螺旋产生相反的效果（8-甲氧基补骨脂素（8-MOP）、安曲霉素和CC-1065加合物）。我们构建了携带一定数量上述每种加合物的质粒DNA，并确定了它们螯合人无细胞提取物中NER因子的潜力。为此，我们测量了受损质粒与定点NER底物切除修复竞争的能力。这种新方法显示，使螺旋不稳定的加合物和使螺旋稳定的加合物螯合NER因子的效率相差超过3个数量级。例如，AAF修饰与NER底物竞争的效率比8-MOP加合物高约1740倍。螯合能力按以下加合物顺序降低：AAF＞紫外线＞/=BPDE＞8-MOP＞安曲霉素、CC-1065。通过监测在特定位点与AAF或CC-1065形成的NER斑块，证实了对使螺旋不稳定损伤的强烈偏好。基于因子螯合和修复合成的这种比较表明，人类NER主要靶向DNA二级结构不稳定的位点。因此，DNA损伤识别的早期步骤涉及对双链体的热力学探测。
