Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATPases Associated with Diverse Cellular Activities
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Adaptor Proteins, Signal Transducing*
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Adenosine Triphosphatases
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Amino Acid Sequence
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Animals
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Base Sequence
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism*
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Cell Line
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Chromatography, Affinity
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DNA Helicases
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DNA Repair
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DNA-Binding Proteins / metabolism*
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Embryo, Mammalian
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Embryo, Nonmammalian
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Fibroblasts
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Fungal Proteins / chemistry
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Fungal Proteins / metabolism*
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Gene Library
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins
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LIM Domain Proteins
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Mice
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Mice, Inbred Strains
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Oligodeoxyribonucleotides
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Peptide Hydrolases / chemistry
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Peptide Hydrolases / metabolism*
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Proteasome Endopeptidase Complex*
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Sequence Alignment
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Sequence Homology, Amino Acid
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Sequence Tagged Sites
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Spodoptera
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TATA-Binding Protein Associated Factors*
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Transcription Factor TFIID*
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Transcription Factor TFIIH
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Transcription Factors / isolation & purification
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Transcription Factors / metabolism*
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Transcription Factors, TFII*
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Transcription, Genetic
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Transcriptional Activation
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Transfection
Substances
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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DNA-Binding Proteins
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Fungal Proteins
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Intracellular Signaling Peptides and Proteins
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LIM Domain Proteins
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Oligodeoxyribonucleotides
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PSMC5 protein, human
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Psmc5 protein, mouse
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Recombinant Proteins
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Repressor Proteins
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SUG1 protein, mammalian
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Saccharomyces cerevisiae Proteins
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TAF6 protein, S cerevisiae
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TATA-Binding Protein Associated Factors
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Transcription Factor TFIID
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Transcription Factors
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Transcription Factors, TFII
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XPBC-ERCC-3 protein
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Transcription Factor TFIIH
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Peptide Hydrolases
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Proteasome Endopeptidase Complex
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ATP dependent 26S protease
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Adenosine Triphosphatases
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RPT6 protein, S cerevisiae
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ATPases Associated with Diverse Cellular Activities
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DNA Helicases