Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination
Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination
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DOI：
10.1021/bi051936t
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发表时间：
2006-02-14
期刊：
影响因子：
2.9
通讯作者：
Turchi, JJ
中科院分区：
文献类型：
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作者：
Trego, KS;Turchi, JJ
XPC-hHR 23 B复合物（XPC-hHR 23 B）是一种异源二聚体蛋白，是全局核苷酸切除修复（NER）途径中DNA损伤识别的初始步骤所需的。XPC-hHR 23 B对UV和顺铂损伤的DNA的强烈偏好先前已使用平衡结合测定法证明。为了更好地理解XPC-hHR 23 B损伤识别的分子机制，我们使用停流荧光测定法对XPC-hHR 23 B-DNA相互作用进行了预稳态动力学分析。XPC-hHR 23 B对顺铂和UV损伤的双链体DNA显示出比未损伤DNA更快的k（on），对k（off）速率具有额外的微小影响。与双链体DNA相比，XPC-hHR 23 B对未受损的单链DNA具有高亲和力，这在很大程度上归因于高缔合率。然而，顺铂对单链DNA的损伤使结合的总体水平降低了7倍，k（on）和k（off）速率的变化几乎具有相等的贡献。总之，这些结果支持XPC-hHR 23 B的初始损伤识别模型，该模型依赖于DNA的结构变化，而不是加合物化学。
The XPC-hHR23B complex (XPC-hHR23B) is a heterodimeric protein required for the initial step of DNA damage recognition in the global nucleotide excision repair (NER) pathway. A strong preference of XPC-hHR23B for UV- and cisplatin-damaged DNA has previously been demonstrated using equilibrium binding assays. To better understand the molecular mechanism of damage recognition by XPC-hHR23B, we carried out the pre-steady-state kinetic analysis of the XPC-hHR23B-DNA interactions using a stopped-flow fluorescence assay. XPC-hHR23B displays a faster k(on) for cisplatin- and UV-damaged duplex DNA than for undamaged DNA, with additional, minor effects on the k(off) rates. XPC-hHR23B has a high affinity for undamaged single-stranded DNA compared to duplex DNA, which can be largely attributed to a high rate of association. However, cisplatin damage on single-stranded DNA reduced the overall level of binding by a factor of 7, with nearly equal contributions from changes to the k(on) and k(off) rates. Together, these results support a model for initial damage recognition by XPC-hHR23B that is dependent on structural changes in the DNA, and not adduct chemistry.