Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, 920-1192, Japan; Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, NC, 27599, USA.
Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, 920-1192, Japan.
Biochem Biophys Res Commun. 2019 Oct 29;519(1):204-210. doi: 10.1016/j.bbrc.2019.08.147. Epub 2019 Sep 4.
The ERCC1-XPF heterodimer is a structure-specific endonuclease and plays multiple roles in various DNA repair pathways including nucleotide excision repair and also telomere maintenance. The dimer formation, which is mediated by their C-terminal helix-hairpin-helix regions, is essential for their endonuclease activity as well as the stability of each protein. However, the detailed mechanism of how a cellular level of ERCC1-XPF is regulated still remains elusive. Here, we report the identification of DDB1- and CUL4-associated factor 7 (DCAF7, also known as WDR68/HAN11) as a novel interacting protein of ERCC1-XPF by mass spectrometry after tandem purification. Immunoprecipitation experiments confirmed their interaction and suggested dominant association of DCAF7 with XPF but not ERCC1. Interestingly, siRNA-mediated knockdown of DCAF7, but not DDB1, attenuated the cellular level of ERCC1-XPF, which is partly dependent on proteasome. The depletion of TCP1α, one of components of the molecular chaperon TRiC/CCT known to interact with DCAF7 and promote its folding, also reduced ERCC1-XPF level. Finally, we show that the depletion of DCAF7 causes inefficient repair of UV-induced (6-4) photoproducts, which can be rescued by ectopic overexpression of XPF or ERCC1-XPF. Altogether, our results strongly suggest that DCAF7 is a novel regulator of ERCC1-XPF protein level and cellular nucleotide excision repair activity.
ERCC1-XPF 异二聚体是一种结构特异性内切酶,在包括核苷酸切除修复在内的多种 DNA 修复途径中发挥多种作用,同时也参与端粒维持。二聚体的形成是由其 C 端螺旋-发夹-螺旋区域介导的,对于其内切酶活性以及每种蛋白质的稳定性都是必不可少的。然而,细胞水平上 ERCC1-XPF 如何被调节的详细机制仍然难以捉摸。在这里,我们通过串联纯化后的质谱分析,鉴定出 DDB1 和 CUL4 相关因子 7(DCAF7,也称为 WDR68/HAN11)是 ERCC1-XPF 的一种新的相互作用蛋白。免疫沉淀实验证实了它们的相互作用,并表明 DCAF7 与 XPF 而不是 ERCC1 主要结合。有趣的是,siRNA 介导的 DCAF7 敲低而非 DDB1 敲低会减弱细胞水平的 ERCC1-XPF,这部分依赖于蛋白酶体。TRiC/CCT 分子伴侣的一个组成部分 TCP1α 的消耗,已知与 DCAF7 相互作用并促进其折叠,也会降低 ERCC1-XPF 水平。最后,我们表明 DCAF7 的耗竭会导致 UV 诱导的(6-4)光产物修复效率降低,而 XPF 或 ERCC1-XPF 的异位过表达可以挽救这一现象。总之,我们的结果强烈表明 DCAF7 是 ERCC1-XPF 蛋白水平和细胞核苷酸切除修复活性的一种新的调节因子。