Recognition of DNA adducts by human nucleotide excision repair - Evidence for a thermodynamic probing mechanism
Recognition of DNA adducts by human nucleotide excision repair - Evidence for a thermodynamic probing mechanism
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DOI：
10.1074/jbc.271.41.25089
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发表时间：
1996-10-11
影响因子：
4.8
通讯作者：
Naegeli, H
中科院分区：
文献类型：
--
作者：
Gunz, D;Hess, MT;Naegeli, H
哺乳动物核苷酸切除修复(NER)检测广泛碱基损伤的机制尚不清楚，在这里，我们测试了人类NER识别笨重修饰的能力，这些修饰要么破坏DNA双螺旋(乙酰氨基荧和苯并[a]芘二醇-环氧化物(BPDE)加合物、紫外线辐射产物)的稳定，要么通过稳定双螺旋(8-甲氧补骨脂素(8-MOP)、蒽霉素和CC-1065加合物)产生相反的作用。我们构建了携带一定数量的这些加合物的质粒DNA，并确定了它们隔离包含在人类无细胞提取物中的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。这一新的方法在螺旋不稳定和螺旋稳定的加合物隔离NER因子的效率上显示出超过3个数量级的差异。例如，AAF修饰能够比8-MOP加合物更有效地与NER底物竞争1740倍，固定能力按以下加合物的顺序递减，AAF&>UV大于或等于BPDE&>8-MOP&>；炭霉素，CC-1065。通过监测AAF或CC-1065在位点特异性加合物上形成的NER补丁，证实了对螺旋破坏稳定损伤的强烈偏好。这种基于因子隔离和修复合成的比较表明，人类NER主要针对DNA二级结构不稳定的部位。因此，DNA损伤识别的早期步骤包括对双链的热力学探测。
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 similar to 1740 times more effectively than 8-MOP adducts, The sequestration potency decreased with the following order of adducts, AAF > UV greater than or equal to 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.