The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay
The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay
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DOI：
10.1021/bi012202t
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发表时间：
2002-05-28
期刊：
影响因子：
2.9
通讯作者：
Krauss, G
中科院分区：
文献类型：
--
作者：
Hey, T;Lipps, G;Krauss, G
XPC-HR23B复合体是全球基因组核苷酸切除修复过程中最初的损伤识别步骤的主要候选者。最近的工作通过电泳迁移率改变分析或免疫沉淀证明了XPC-HR23B复合体与各种类型的受损DNA底物之间的特异性相互作用。虽然这些研究允许估计不同类型病变的相对结合亲和力，但大量竞争DNA的存在或戊二醛固定的需要阻碍了平衡常数的量化。我们利用荧光各向异性测量对XPC与受损DNA的结合进行了定量研究。XPC-HR23B复合体以高亲和力(K-D类似于1-3 nm)与含有单个顺铂1，3-链内加合物或六核苷酸错配区的36个核苷酸荧光DNA片段结合。化学计量滴定实验表明，XPC-HR23B制剂具有接近70%的DNA结合活性。使用荧光探针与单一确定的光产物结合的实验表明，与环丁烷二聚体相比，XPC对6，4-光产物的偏好是30倍。与未损伤和损伤的质粒DNA的竞争实验表明，XPC-HR23B复合体区分损伤和未损伤的部位具有很高的特异性。特异性系数介于100和3000之间，具体取决于计算中考虑的非特定部位的数量。当XPA加入到XPC结合反应混合物中时，不可能在带有铂的36个碱基的探针上检测到协同三元络合物的形成。
The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K-D similar to 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that similar to70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.