Weeda G, Rossignol M, Fraser R A, Winkler G S, Vermeulen W, van 't Veer L J, Ma L, Hoeijmakers J H, Egly J M
Department of Cell Biology, Medical Genetics Center, Erasmus University, Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Nucleic Acids Res. 1997 Jun 15;25(12):2274-83. doi: 10.1093/nar/25.12.2274.
Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation.
基础转录起始/DNA修复因子TFIIH的突变可导致三种人类疾病:着色性干皮病(XP)、科凯恩综合征(CS)和毛发硫营养不良(TTD)。CS和TTD的非修复特征被认为是由于该复合物转录功能的部分失活所致。为了寻找其与TFIIH亚基的相互作用因患者突变而受到干扰的蛋白质,我们使用酵母双杂交系统,并报告了一种新型的与XPB相互作用的蛋白质SUG1的分离。这种相互作用通过以下方式在体内和体外得到验证。(i)在双杂交试验中,SUG1与XPB相互作用,但不与其他核心TFIIH亚基相互作用。(ii)在杆状病毒共表达系统中观察到物理相互作用。(iii)在非过表达条件下的成纤维细胞中,通过使用功能性标记的TFIIH进行共纯化、免疫纯化和镍螯合亲和色谱法推断,一部分SUG1与TFIIH全复合物结合。此外,正常成纤维细胞中SUG1的过表达在体内诱导转录停滞和染色质塌陷。有趣的是,与XPB的突变形式的相互作用减弱,从而为XP-B患者的临床特征提供了潜在联系。由于SUG1是26S蛋白酶体的一个组成部分,可能是中介体的一部分,我们的发现揭示了TFIIH与参与蛋白质建模/降解的细胞机制之间的SUG1依赖性联系。