Program in Molecular Biophysics and Structural Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA.
UPMC Hillman Cancer Center, Pittsburgh, PA, 15213, USA.
Nat Commun. 2020 Mar 13;11(1):1356. doi: 10.1038/s41467-020-15168-1.
Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.
核苷酸切除修复（NER）可去除多种 DNA 损伤，包括 UV 诱导的光产物和大体积碱基加合物。XPA 是真核 NER 中的必需蛋白，但有关其化学计量和损伤识别作用的报道存在争议。本文通过 PeakForce Tapping 原子力显微镜，发现人源 XPA 作为单体结合并使 DNA 弯曲约 60°。此外，我们观察到 XPA 对螺旋扭曲碱基加合物 N-(2'-脱氧鸟嘌呤-8-基)-2-乙酰氨基芴的特异性，而非损伤 dsDNA。此外，单分子荧光显微镜显示，与 DNA 结合的 XPA 在暂停阶段之间表现出多种线性扩散模式。DNA 损伤的存在增加了暂停的频率。缺乏固有无序的 N 端和 C 端的截短 XPA，丧失了对 DNA 损伤的特异性，在损伤 DNA 上的暂停减少。我们的数据与一个工作模型一致，即单体 XPA 使 DNA 弯曲，沿 DNA 表现出间歇性的线性扩散，并在 DNA 损伤时暂停。
